Molecular detection systems utilizing reiterative oligonucleotide synthesis

ABSTRACT

The present invention provides methods for detecting the presence of a target molecule by generating multiple detectable oligonucleotides through reiterative enzymatic oligonucleotide synthesis events on a defined polynucleotide sequence. The methods generally comprise using a nucleoside, a mononucleotide, an oligonucleotide, or a polynucleotide, or analog thereof, to initiate synthesis of an oligonucleotide product that is substantially complementary to a target site on the defined polynucleotide sequence; optionally using nucleotides or nucleotide analogs as oligonucleotide chain elongators; using a chain terminator to terminate the polymerization reaction; and detecting multiple oligonucleotide products that have been synthesized by the polymerase. In one aspect, the invention provides a method for detecting a target protein, DNA or RNA by generating multiple detectable RNA oligoribonucleotides by abortive transcription.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to the detection and kitsfor the detection of target molecules and, more particularly, to nucleicacid-based detection assays that produce multiple signals from a targetmolecule by generating multiple copies of detectable oligonucleotidesthrough reiterative synthesis events on a defined nucleic acid template,particularly via abortive transcription initiation. The method and kitsof the invention may be used to detect mutations, RNA molecules,pathogens, proteins, or pre-cancerous conditions.

[0003] 2. Related Art

[0004] The development of various methods for nucleic acid detection andthe detection of nucleic acid amplification products has led to advancesin the detection, identification, and quantification of nucleic acidsequences in recent years. Nucleic acid detection is potentially usefulfor both qualitative analyses, such as the detection of the presence ofdefined nucleic acid sequences, and quantitative analyses, such as thequantification of defined nucleic acid sequences. For example, nucleicacid detection may be used to detect and identify pathogens; detectgenetic and epigenetic alterations that are linked to definedphenotypes; diagnose genetic diseases or the genetic susceptibility to aparticular disease; assess gene expression during development, disease,and/or in response to defined stimuli, including drugs; as well asgenerally foster advancements in the art by providing researchscientists with additional means to study the molecular and biochemicalmechanisms that underpin cellular activity.

[0005] Nucleic acid detection technology generally permits the detectionof defined nucleic acid sequences through probe hybridization, that is,the base-pairing of one nucleic acid strand with a second strand of acomplementary, or nearly complementary, nucleic acid sequence to form astable, double-stranded hybrid. Such hybrids may be formed of aribonucleic acid (RNA) segment and a deoxyribonucleic acid (DNA)segment, two RNA segments, or two DNA segments, provided that the twosegments have complementary or nearly complementary nucleotidesequences. Under sufficiently stringent conditions, nucleic acidhybridization may be highly specific, requiring exact complementaritybetween the hybridized strands. Typically, nucleic acid hybrids comprisea hybridized region of about eight or more base pairs to ensure thebinding stability of the base-paired nucleic acid strands. Hybridizationtechnology permits the use of one nucleic acid segment, which isappropriately modified to enable detection, to “probe” for and detect asecond, complementary nucleic acid segment with both sensitivity andspecificity. In the basic nucleic acid hybridization assay, asingle-stranded target nucleic acid (either DNA or RNA) is hybridized,directly or indirectly, to a labeled nucleic acid probe, and theduplexes containing the label are quantified. Both radioactive andnon-radioactive labels have been used.

[0006] However, a recognized disadvantage associated with nucleic acidprobe technology is the lack of sensitivity of such assays when thetarget sequence is present in low copy number or dilute concentration ina test sample. In many cases, the presence of only a minute quantity ofa target nucleic acid must be accurately detected from among myriadother nucleic acids that may be present in the sample. The sensitivityof a detection assay depends upon several factors: the ability of aprobe to bind to a target molecule; the magnitude of the signal that isgenerated by each hybridized probe; and the time period available fordetection.

[0007] Several methods have been advanced as suitable means fordetecting the presence of low levels of a target nucleic acid in a testsample. One category of such methods is generally referred to as targetamplification, which generates multiple copies of the target sequence,and these copies are then subject to further analysis, such as by gelelectrophoresis, for example. Other methods generate multiple productsfrom a hybridized probe, or probes, by, for example, cleaving thehybridized probe to form multiple products or ligating adjacent probesto form a unique, hybridization-dependent product. Still other methodsamplify signals generated by the hybridization event, such as a methodbased upon the hybridization of branched DNA probes that have a targetsequence binding domain and a labeled reporting sequence binding domain.

[0008] There are many variations of target nucleic acid amplification,including, for example, exponential amplification, ligation-basedamplification, and transcription-based amplification. An example of anexponential nucleic acid amplification method is the polymerase chainreaction (PCR), which has been disclosed in numerous publications. See,for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Mullis et al. U.S. Pat. No. 4,582,788; and Saiki R.et al. U.S. Pat. No. 4,683,194. An example of ligation-basedamplification is the ligation amplification reaction (LAR) which isdisclosed by Wu et al. in Genomics 4:560 (1989). Various methods fortranscription-based amplification are disclosed in U.S. Pat. Nos.5,766,849 and 5,654,142; and also in Kwoh et al., Proc. Natl. Acad. Sci.U.S.A. 86:1173 (1989).

[0009] The most commonly used target amplification method is thepolymerase chain reaction (PCR), which consists of repeated cycles ofDNA polymerase-generated primer extension reactions. Each reaction cycleincludes heat denaturation of the target nucleic acid; hybridization tothe target nucleic acid of two oligonucleotide primers, which bracketthe target sequence on opposite strands of the target that is to beamplified; and extension of the oligonucleotide primers by a nucleotidepolymerase to produce multiple, double-stranded copies of the targetsequence. Many variations of PCR have been described, and the method isbeing used for the amplification of DNA or RNA sequences, sequencing,mutation analysis, and others. Thermocycling-based methods that employ asingle primer have also been described. See, for example, U.S. Pat. Nos.5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. The primercan be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No.5,744,308. Other methods that are dependent on thermal cycling are theligase chain reaction (LCR) and the related repair chain reaction (RCR).

[0010] Target nucleic acid amplification may be carried out throughmultiple cycles of incubation at various temperatures (i.e., thermalcycling) or at a constant temperature (i.e., an isothermal process). Thediscovery of thermostable nucleic acid modifying enzymes has contributedto rapid advances in nucleic acid amplification technology. See, Saikiet al., Science 239:487 (1988). Thermostable nucleic acid modifyingenzymes, such as DNA and RNA polymerases, ligases, nucleases, and thelike, are used in methods that are dependent on thermal cycling as wellas in isothermal amplification methods.

[0011] Isothermal methods, such as strand displacement amplification(SDA) for example, are disclosed by Fraiser et al. in U.S. Pat. No.5,648,211; Cleuziat et al. in U.S. Pat. No. 5,824,517; and Walker etal., Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992). Other isothermaltarget amplification methods include transcription-based amplificationmethods in which an RNA polymerase promoter sequence is incorporatedinto primer extension products at an early stage of the amplification(WO 89/01050), and a further, complementary, target sequence isamplified through reverse transcription followed by physical separationor digestion of an RNA strand in a DNA/RNA hybrid intermediate product.See, for example, U.S. Pat. Nos. 5,169,766 and 4,786,600. Furtherexamples of transcription-based amplification methods includeTranscription Mediated Amplification (TMA), Self-Sustained SequenceReplication (3SR), Nucleic Acid Sequence Based Amplification (NASBA),and variations there of. See, for example, Guatelli et al. Proc. Natl.Acad. Sci. U.S.A. 87:1874-1878 (1990) (3SR); U.S. Pat. No. 5,766,849(TMA); and U.S. Pat. No. 5,654,142 (NASBA).

[0012] These and other techniques have been developed recently to meetthe demands for rapid and accurate detection of pathogens, such asbacteria, viruses, and fungi, for example, as well as the detection ofnormal and abnormal genes. While all of these techniques offer powerfultools for the detection and identification of minute amounts of a targetnucleic acid in a sample, they all suffer from various problems.

[0013] One problem, especially for PCR, is that conditions foramplifying the target nucleic acid for subsequent detection and optionalquantitation vary with each test, that is, there are no constantconditions favoring test standardization. Further, amplification methodsthat use a thermocycling process have the added disadvantage of extendedlag times which are required for the thermocycling block to reach the“target” temperature for each cycle. Consequently, amplificationreactions performed using thermocycling processes require a significantamount of time to reach completion. The various isothermal targetamplification methods do not require a thermocycler and are thereforeeasier to adapt to common instrumentation platforms. However, thepreviously described isothermal target amplification methods also haveseveral drawbacks. Amplification according to the SDA methods requiresthe presence of defined sites for restriction enzymes, which limits itsapplicability. The transcription-based amplification methods, such asthe NASBA and TMA methods, are limited by the need to incorporate apolymerase promoter sequence into the amplification product by a primer.

[0014] Accordingly, there is a need for rapid, sensitive, andstandardized nucleic acid signal detection methods that can detect lowlevels of a target nucleic acid in a test sample. These needs, as wellas others, are met by the inventions of this application.

[0015] All patents, patent publications, and scientific articles citedor identified in this application are hereby incorporated by referencein their entirety to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by referencein its entirety.

SUMMARY OF THE INVENTION

[0016] The invention provides methods and compositions for producingmultiple detectable signals through reiterative oligonucleotidesynthesis reactions on a defined polynucleotide for the detection oftarget molecules. The invention also provides applications for thereiterative synthesis and detection methods. Important applications ofthe methods and kits of the invention, include but are not limited todetection of mutations and single nucleotide polymorphisms, RNAmolecules, pathogens, and detection of pre-cancerous or cancerousmutations and conditions.

[0017] Accordingly, in one aspect, the invention provides a method forsynthesizing multiple complementary oligonucleotides from a target DNAor RNA polynucleotide. The method comprises the steps of: (a)hybridizing an initiator (nucleoside, mononucleotide, oligonucleotide orpolynucleotide) with a single-stranded target polynucleotide (RNA orDNA); (b) incubating said target polynucleotide and initiator with anRNA-polymerase, a terminator, and optionally additional ribonucleotides;(c) synthesizing multiple oligonucleotides from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotides, thereby synthesizingmultiple reiterative oligonucleotides.

[0018] In another aspect, the invention provides a method for detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of (a) hybridizing aninitiator with a single stranded target polynucleotide; (b) incubatingsaid target polynucleotide and initiator with an RNA-polymerase, aterminator and optionally additional ribonucleotides; (c) synthesizingmultiple oligonucleotides from said target polynucleotide, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple reiterativeoligonucleotides; and (d) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts of a polynucleotide of interest.

[0019] In a further aspect, the invention provides a method of detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of: (a) hybridizing aninitiator to a single-stranded target polynucleotide; (b) incubatingsaid target polynucleotide and initiator with a target site probe, anRNA-polymerase, a terminator and optionally additional ribonucleotides,wherein said target site probe hybridizes with said targetpolynucleotide; (c) synthesizing an oligonucleotide transcript that iscomplementary to said target site from said target polynucleotide,wherein said initiator is extended until said terminator is incorporatedinto said oligonucleotide transcript, thereby synthesizing multiplereiterative oligonucleotide transcripts; and (d) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts,wherein said oligonucleotides being synthesized are one of the lengthsselected from the group consisting of: about 2 to about 26 nucleotides,about 26 to about 50 nucleotides and about 50 nucleotides to about 100nucleotides, and greater than 100 nucleotides.

[0020] In a further aspect, the invention provides a method fordetecting methylated cytosine residues at CpG sites in a targetpolynucleotide. The method comprises the steps of: (a) deaminating asingle-stranded target DNA sequence under conditions which convertunmethylated cytosine residues to uracil residues while not convertingmethylated cytosine residues to uracil; (b) hybridizing an initiatorwith a single stranded target polynucleotide; (c) incubating saiddeaminated target polynucleotide and said initiator with a terminator,an RNA-polymerase and optionally additional ribonucleotides, wherein atleast one of said initiator, terminator, or optional ribonucleotides ismodified to enable detection of hybridization to the CG sites; (d)synthesizing an oligonucleotide transcript that is complementary to saidCG sites from said target polynucleotide, wherein said initiator isextended until said terminator is incorporated into said oligonucleotidetranscript thereby synthesizing multiple reiterative oligonucleotidetranscripts; and (e) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts.

[0021] In still a further aspect, the invention provides a method fordetecting methylated cytosine residues at a CpG site in a target gene.The method comprises the steps of: (a) deaminating a single-strandedtarget DNA polynucleotide under conditions which convert unmethylatedcytosine residues to uracil residues while not converting methylatedcytosine residues to uracil; (b) hybridizing a target site probe withsaid single stranded target polynucleotide; (c) incubating said targetpolynucleotide and target site probe with, an initiator, a terminator,an RNA-polymerase, and optionally additional ribonucleotides, whereinsaid at least one of said initiator, said terminator or said nucleotidesare complementary to the CpG site; (d) synthesizing an oligonucleotidetranscript that is complementary to said target site from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotides, thereby synthesizingmultiple reiterative oligonucleotide transcripts; and (e) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts.

[0022] In still a further aspect, the invention provides a method fordetecting the presence or absence of mutations in a target DNA sequence.The method comprises the steps of: (a) hybridizing a target site probeto a single-stranded DNA polynucleotide, wherein said DNA polynucleotidemay contain a mutation relative to a normal or wild type gene; (b)incubating said target polynucleotide and target-site probe with anRNA-polymerase, a initiator, a terminator and optionally additionalribonucleotides; (c) synthesizing an oligonucleotide transcript fromsaid target polynucleotide that is complementary to a target mutationsite, wherein said initiator is extended until said terminator isincorporated into said oligonucleotides thereby synthesizing multipleabortive reiterative oligonucleotides; and (d) determining the presenceor absence of a mutation by detecting or quantifying said reiterativelysynthesized oligonucleotides transcribed from said target DNApolynucleotide.

[0023] In another aspect, the invention provides a method for detectingmutations in a target DNA polynucleotide using a capture probe. Themethod comprises the steps of: (a) immobilizing a capture probe designedto hybridize with said target DNA polynucleotide; (b hybridizing saidcapture probe to said target DNA polynucleotide, wherein said DNApolynucleotide may contain a mutation relative to a normal or wild typegene; (c) incubating said target polynucleotide and with anRNA-polymerase, initiator, a terminator and optionally additionalribonucleotides; (d) synthesizing an oligonucleotide transcript that iscomplementary to a target site from said target polynucleotide, whereinsaid initiator is extended until said terminator is incorporated intosaid oligonucleotide transcript, thereby synthesizing multiple abortivereiterative oligonucleotide transcripts; and (e) determining thepresence or absence of a mutation by detecting or quantifying saidreiteratively synthesized oligonucleotide transcripts from said targetDNA polynucleotide.

[0024] In another aspect, the invention provides a method for detectingDNA or RNA in a test sample. The method comprises the steps of: (a)hybridizing a single stranded target polynucleotide with an abortivepromoter cassette comprising a sequence that hybridizes to the singlestranded target polynucleotide, and a region that can be detected bytranscription by a polymerase; (b) incubating said target polynucleotidewith an RNA-polymerase, an initiator, a terminator and optionallyadditional ribonucleotides; (c) synthesizing an oligonucleotidetranscript that is complementary to the initiation start site of theAPC, wherein said initiator is extended until said terminator isincorporated into said oligonucleotides, thereby synthesizing multiplereiterative oligonucleotide transcripts; and (d) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts.

[0025] In another aspect, the invention provides a method for detectingthe presence of pathogens in a test sample. The method comprises thesteps of: (a) hybridizing a single stranded target pathogenpolynucleotide in said test sample with an abortive promoter cassettecomprising a region that can be detected by transcription by apolymerase; (b) incubating said target polynucleotide and initiator withan RNA-polymerase, a terminator and optionally additionalribonucleotides; (c) synthesizing an oligonucleotide transcript that iscomplementary to initiation start site of the APC, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple abortive reiterativeoligonucleotide transcripts; and (d) determining the presence of apathogen by detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts synthesized from said test sample.

[0026] In still a further aspect, the invention provides a method fordetecting pathogens in a test sample using a capture probe. The methodcomprises the steps of: (a) immobilizing a capture probe designed tohybridize with a target DNA polynucleotide in said test sample; (b)hybridizing said capture probe with a test sample that potentiallycontains said target DNA polynucleotide; (c) hybridizing a singlestranded target DNA polynucleotide in said test sample with an abortivepromoter cassette comprising a region that hybridizes to the singlestranded target pathogen polynucleotide, and a region that can bedetected by transcription by a polymerase; (d) incubating said targetpolynucleotide with an RNA-polymerase, initiator, a terminator andoptionally additional ribonucleotides; (e) synthesizing anoligonucleotide transcript that is complementary to said initiationtranscription start site of APC, wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotides therebysynthesizing multiple reiterative oligonucleotide transcripts; and (f)determining the presence or absence of a pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcripts.

[0027] In still a further aspect, the invention provides a method fordetecting mRNA expression in a test sample. The method comprises thesteps of: (a) hybridizing a target mRNA sequence with an abortivepromoter cassette comprising a region that can be detected bytranscription by a polymerase; (b) incubating said target mRNA sequencewith an RNA-polymerase, an initiator, a terminator and optionallyadditional ribonucleotides; (c) synthesizing an oligonucleotidetranscript that is complementary to transcription initiation start site,wherein said initiator is extended until said terminator is incorporatedinto said oligonucleotide transcript, thereby synthesizing multiplereiterative oligonucleotides; and (d) determining the presence orabsence of the mRNA by detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts synthesized from said testsample.

[0028] In still a further aspect, the invention provides a method fordetecting an oligonucleotide synthesized from a target DNA sequence. Themethod comprises the steps of: (a) hybridizing a DNA primer with asingle-stranded target DNA sequence; (b) extending said DNA primer witha DNA polymerase and nucleotides, such that said DNA polymerasereiteratively synthesizes a nucleotide sequence; and (c) detectingoligonucleotide comprised of repeat sequences synthesized by said DNApolymerase.

[0029] In still a further aspect, the invention provides a method forproducing a microarray. The method comprises the steps of: (a)synthesizing multiple abortive oligonucleotide replicates from a targetDNA sequence by the method of claim 1; and (b) attaching said multipleabortive oligonucleotide replicates to a solid substrate to produce amicroarray of said multiple abortive oligonucleotide replicates.

[0030] In still a further aspect, the invention provides a method fordetecting multiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of: (a) incubating asingle-stranded target polynucleotide in a mixture comprising aninitiator, an RNA-polymerase and optionally additional ribonucleotides;(b) synthesizing multiple oligonucleotide transcripts from said targetpolynucleotide, wherein said initiator is extended until terminated dueto nucleotide deprivation, thereby synthesizing multiple abortivereiterative oligonucleotide transcripts; and (c) detecting orquantifying said reiteratively synthesized oligonucleotides.

[0031] In still a further aspect, the invention provides a method ofdetecting multiple reiterated oligonucleotides from a target DNA or RNApolynucleotide with a target site probe. The method comprises the stepsof: (a) incubating a single-stranded target polynucleotide in a mixturecomprising an initiator, an RNA-polymerase, a target site probe andoptionally additional ribonucleotides, wherein said target site probeand said target polynucleotide hybridize to form a bubble complexcomprising a first double-stranded region upstream of a target site, asingle-stranded region comprising said target site, and a seconddouble-stranded region downstream of said target site; (b) synthesizingmultiple oligonucleotide transcripts from said target polynucleotide,wherein said initiator is extended until terminated due to nucleotidedeprivation, thereby synthesizing multiple abortive reiterativeoligonucleotides; and (c) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts.

[0032] In still a further aspect, the invention provides a method fordetecting methylated cytosine residues at a CG site near a target gene.The method comprises the steps of:

[0033] (a) deaminating a single-stranded target DNA sequence underconditions which convert unmethylated cytosine residues to uracilresidues while not converting methylated cytosine residues to uracil;(b) incubating a single-stranded target polynucleotide in a mixturecomprising an initiator, a terminator, an RNA-polymerase, a target siteprobe and optionally additional ribonucleotides; (c) synthesizingmultiple oligonucleotide transcripts from said target polynucleotide,wherein said initiator is extended until terminated due to nucleotidedeprivation, thereby synthesizing multiple abortive reiterativeoligonucleotide transcripts; and (d) detecting or quantifying saidreiteratively synthesized oligonucleotides.

[0034] In still a further aspect, the invention provides a method fordetecting a target protein in a test sample. The method comprises thesteps of: (a) covalently attaching the target protein to an abortivepromoter cassette (APC) by a reactive APC linker, wherein said APCcomprises a region that can be detected by transcription by apolymerase; (b) incubating said target protein with an RNA-polymerase,an initiator, a terminator and optionally additional ribonucleotides;(c) synthesizing an oligonucleotide transcript that is complementary totranscription initiation start site of APC, wherein said initiator isextended until said terminator is incorporated into said oligonucleotidetranscript, thereby synthesizing multiple reiterative oligonucleotidetranscripts; and (d) determining the presence or absence of the targetprotein by detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts synthesized from said test sample.

[0035] In still a further aspect, the invention provides a method fordetecting cancer. The method comprises the steps of: (a) obtaining atissue sample from a patient in need of detection of a cancer; (b)deaminating the DNA under conditions which convert unmethylated cytosineresidues to uracil residues while leaving the methylated cytosineresidues unaltered; (c) hybridizing an initiator to a targetpolynucleotide wherein said initiator is a mononucleoside,mononucleotide, binucleotide, oligonucleotide, polynucleotide, or ananalog thereof; (d) incubating said deaminated target polynucleotide andsaid initiator with a terminator, an RNA-polymerase and optionallyadditional ribonucleotides, wherein at least one of said initiator,terminator, or optional ribonucleotides is modified to enable detectionof hybridization to the CG sites; (e) synthesizing an oligonucleotidetranscript that is complementary to said CG sites from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotide transcript therebysynthesizing multiple reiterative oligonucleotide transcripts; (f)detecting or quantifying said reiteratively synthesized oligonucleotidetranscripts; and (g) comparing the results with those obtained similarlyfrom a control sample.

[0036] In still a further aspect, the invention provides a method fordetecting pathogens. The method comprises the steps of: (a) obtaining asample in need of detection of a pathogen; (b) hybridizing a singlestranded target pathogen polynucleotide in said sample with an abortivepromoter cassette comprising a nucleotide sequence that hybridizes tosingle stranded target pathogen polynucleotide, and a region that can bedetected by transcription by a polymerase; (c) incubating said targetpolynucleotide and initiator with an RNA-polymerase, a terminator andoptionally additional ribonucleotides; (d) synthesizing anoligonucleotide transcript that is complementary to initiation startsite of the APC, wherein said initiator is extended until saidterminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (e) determining the presence of a pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said test sample.

[0037] The present invention also provides kits for conducting theoligonucleotide synthesis and detection methods described herein. In oneaspect, for example, the invention provides reagent containers, whichcontain various combinations of the components described herein. Thesekits, in suitable packaging and generally (but not necessarily)containing suitable instructions, contain one or more components used inthe oligonucleotide synthesis and detection methods. The kit may alsocontain one or more of the following items: polymerization enzymes,initiators, primers, buffers, nucleotides, control DNA, antibodies,streptavidin, and biotin. The kit may also contain reagents mixed inappropriate amounts for performing the methods of the invention. Thereagent containers preferably contain reagents in unit quantities thatobviate measuring steps when performing the subject methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1: Abortive Promoter Cassettes. Abortive Promoter Cassettes(APC) are regions of nucleic acid that form a polymerase binding siteand can be attached to other macromolecules through interaction with aspecific nucleic acid sequence, which is termed the APC linker. APClinkers can be attached to target nucleic acids (DNA or RNA) byhybridization to complementary sequences on either the template ornon-template strands of the target nucleic acid. An APC Linker can alsohybridize to a complementary sequence placed on any target molecule,such as a protein, for detection of molecules that bind to said protein.Multiple detectable oligonucleotides are generated by polymerase boundto the Abortive Promoter Cassette. In this figure, the APC depictedcontains two regions of essential complementarity (A, A′ and C, C′),which are separated by a “bubble region.” In this schematic, the “bubbleregion” is generated because regions of the two strands arenon-complementary (B, and E). Alternatively, the APC may have twocompletely complementary strands. Upon binding of the RNA polymerase,the DNA strands separate, which leads to the formation of the “bubbleregion.”

[0039] Regions A, B, and C are on one strand. Regions C′, E, and A′ areon the complementary strand. The APC may be made from two separatestrands (ABC and C′EA′) or all 6 regions may be on a singlepolynucleotide, in which regions C and C′ are separated by a linkerregion D, which can modified to be as long as needed. Linker region Dmay serve only to join C and C′ or the sequence of region D may serve asa binding site for other factors that may enhance abortivetranscription, such as transcription roadblock proteins, including butnot limited to EcoRI QIII mutant, the lac repressor and other RNApolymerases. The linker region D may be designed for a single road blockprotein, or multiple roadblock proteins. The length of linker region Dwill depend on the function of the linker region.

[0040]FIG. 2: Signal Generation by Reiterative oligonucleotidesynthesis. A signal is generated by the enzymatic incorporation of oneor more nucleotide analogs into multiple (n) highly similar or identicaloligonucleotide products. Under appropriate conditions, RNAoligonucleotides can be made from nucleic acid templates in the absenceof a promoter. An initiator may be comprised of one or more nucleosides,nucleoside analogs, nucleotides, or nucleotide analogs. The initiar maycontain one or more covalently joined nucleotides, including but notlimited to, 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides,76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides,151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250nucleotides and more than 250 nucleotides, and may contain a functionalR group. The initiator (n copies) can be elongated directly with ncopies of a terminator to end chain elongation or n copies of otherelongator nucleotides (Y positions) may be incorporated between theinitiator and the terminator to form a longer oligonucleotide. Theterminator may contain a second functional group. N_(I)=Initiatingmononucleotide or oligonucleotide analog, N_(E)=Elongatingmononucleotides or analog, N_(T)=Terminating mononucleotide or analog,Z=x+y; R₁=H, OH, or reporter group; R₂=H, OH, or reporter group; N=deoxyor ribonucleotides; Polymerase=RNA-dependent or DNA-dependent RNApolymerase. DNA or RNA may be attached to other molecules, such asproteins

[0041]FIG. 3: 5′AEDANS-S-AMP synthesis. Example of a mononucleotidetranscription initiator: IAEDANS (5-((2-((iodoacetyl)amino)ethyl)amino-1-Napthalenesulfonic acid alkylates AMPS(5-α-thio-AMP) to form the fluorescent transcription initiator. Thisanalog can only initiate transcription because it lacks a 5′triphosphate group and can therefore not be incorporated internally orterminally.

[0042]FIG. 4: Nucleotides that can be elongators or terminators.Nucleotide analogs that may be included at internal or 3′ terminalpositions in oligonucleotides are shown. All of these analogs can beconverted to terminators simply by replacement of the 3′ OH group.

[0043]FIG. 5: Other fluorescent groups that may be R₁ or R₂. Theoligonucleotides can be labeled with a variety of functional groups.Several of the preferred fluorescent groups are shown.

[0044]FIG. 6: Dinucleotide synthesis via abortive initiation onsingle-stranded DNA or RNA. Single stranded (ss) nucleic acid is DNA orRNA. Polymerase is a DNA-dependent or RNA-dependent RNA polymerase.N_(I)=3′-OH nucleoside or nucleotide initiator; N_(T)=5′-triphosphatenucleotide or nucleotide analog terminator. R₁ may be on the 5′phosphate group, the 2′ position of the sugar, or on the purine orpyrimidine base. R₂ may be on the pyrimidine or purine base or 2′ or 3′position of the sugar/ribose or deoxyribose. R₁=H, OH, and/or anyreporter group or reporter group precursor, as described herein. R₂=H,OH, and/or any reporter group or reporter group precursor, as describedherein. Signal may be any signal that can be detected, and includes butis not limited to fluorescence, fluorescence resonance energy transfer(FRET), or colorimetric. As one example, R₁ may be AEDANS, and R₂ may beFluorescein. Signal is generated by FRET from R₁ to R₂.

[0045]FIG. 7: 5′-AEDANS-SpApU-FLUORESCEIN. Dinucleotide generated byabortive initiation for FRET detection. When excited by light of theappropriate wavelength, R₁ (AEDANS) donates fluorescent energy to R₂(fluorescein), which then emits fluorescent light of a differentwavelength that can be detected and quantified. This fluorescenceresonance energy transfer (FRET) only occurs when the two groups arejoined together to form AEDANS-SpApU-Fluorescein during transcription,which brings the two groups close enough to each other for efficientenergy transfer.

[0046]FIG. 8: Signal generation via dinucleotide production.Oligonucleotides can be synthesized that contain one R group on theinitiator nucleotide and another on the terminator nucleotide, such thatthe R groups have different functions. For example, if R₁ is biotin, itcan be used for oligonucleotide product immobilization and R₂ allows forsignal production.

[0047] Example 1: R₁ tag=biotin, R₂ tag=fluorescein: detection offluorescein emission

[0048] Example 2: R₁ tag=DNP, R₂ tag=reactive thiol

[0049]FIG. 9: Signal generation for FRET detection by abortiveinitiation. Oligonucleotides can be reiteratively synthesized thatcontain 2 to 25 nucleotides and have two different R groups, one at ornear each end of the oligonucleotide product made during transcription.Energy transfer between the two R groups on the substrates can onlyoccur after they are brought into proximity during template-directedoligonucleotide synthesis by enzymatic phosphodiester bond formationbetween the labeled initiator and the labeled terminator nucleotides.The R₁ donor group on N_(I) can be excited by irradiating the samplewith light of wavelength of λ_(1A), where λ_(1A) is the absorptionmaximum of group R₁. The excited R₁ donor group emits light ofwavelength λ_(1E), where λ_(1E) is the emission maximum for group R₁ andalso a wavelength for absorption by group R₂ (λ_(2A)). The acceptor R₂group on N_(T) absorbs light of wavelength λ_(1E)/λ_(2A) that wasemitted by the excited R₁ donor group on N_(I). The excited acceptor R₂group on N_(T) emits light of wavelength λ_(2E), which is detected andquantified. Similarly, R₂ may be an energy donor to R₁, with emissionfrom R₁ detected. In the absence of target-associated template, nooligonucleotide is synthesized.

[0050]FIG. 10: Trinucleotide energy transfer. Labeled oligonucleotidesynthesis is initiated with a labeled dinucleotide initiator. The labelmay be on either the 5′ nucleotide (R₁) or the 3′ nucleotide (R₂) of thedinucleotide initiator. The initiator is elongated with a labeled (R₃)5′-nucleosidetriphosphate terminator nucleotide analog. Detection viaenergy transfer can be adjusted to utilize R₁ or R₂ with R₃, as shown.In the absence of nucleic acid template-directed phosphodiester bondformation between the initiator and terminator, the R groups remainsufficiently separated that no energy transfer is detected. In thisexample, the amount of energy emitted as λ_(3E) is directly proportionalto the amount of template-associated target present. Similarly, the Rgroups may be varied for other applications, as demonstrated in FIG. 8.

[0051]FIG. 11: Target Site Probe. An RNA polymerase can be directed tospecific nucleotide positions (sites) in target nucleic acids by thehybridization of a gene-specific or region-specific Target Site Probe(TSP). The target site is a nucleotide position in the DNA to beanalyzed for sequence (as in detection of single nucleotidepolymorphisms) or structure (as in assessing the methylation status of aspecific nucleotide), and it is located on the template strand of thetarget sequence at the junction of regions E and C′ in the targetsequence. The TSP contains a region of homology to the target nucleicacid (Region A) which begins approximately 8-14 nucleotides and endsapproximately 15-35 nucleotides upstream of the target site nucleotide.A second region of the TSP is designed to be non-complementary to the8-14 nucleotides immediately upstream of the target site (Region B), sothat a melted “bubble” region forms when the TSP hybridizes to thetarget nucleic acid. The TSP contains a third region (Region C) which isessentially complementary to the 5-25 nucleotides immediately downstreamof the target site nucleotide. RNA polymerase will bind to the bubblecomplex such that transcription will start at the E/C′ junction and willmove downstream into the C/C′ hybrid.

[0052]FIG. 12: Methylation of CpG Islands in DNA. The human genome has a4-5 fold lower frequency of CpG dinucleotides than expected given theoverall frequency of C and G in human DNA. A large fraction of CpGsequence is distributed into clusters known as CpG islands. Thesesequence patterns are between 300-3000 nucleotides long and overlap withabout 60% of all human promoters. The remaining CpG dinucleotidesoutside of CpG islands contain methylated C. CpG methylation outside ofCpG islands stabilize the genome by inactivating the expression ofparasitic DNA, and independently play an essential role in development.Changes in the methylation status of cytosine in CpG islands are earlyevents in many cancers and permanent changes found in many tumors. TheseCpG islands are found in the regions next to genes that determinewhether the gene is “ON” or “OFF”. Many genes that are important forpreventing cancer, such as tumor suppressor genes, need to be “ON” forcells to grow normally. Cellular enzymes can add methyl groups(methylation) to the C residues in these CpG islands. This methylationresults in the shutting “OFF” of these genes. When tumor suppressorgenes are shut “OFF”, the cell no longer makes the proteins that theyencode, and the cell begins to grow without control checkpoints. This isone of the early events that can lead to cell “transformation” and theprogression of cancer.

[0053]FIG. 13: Deamination conversion of unmethylated cytosine groups inDNA. Deamination converts unmethylated C to U. Methylated C groups, suchas those in CpG islands that regulate eukaryotic genes, are resistant todeamination and remain as C in the product DNA. If 100% deaminationoccurs, methylated DNA will still contain CpG doublets, whereasunmethylated DNA will contain no cytosine and will now contain UpG whereCpG doublets were before deamination. This difference in DNA sequencecan be used to distinguish between methylated and unmethylated DNA byabortive transcription because the two DNAs encode differentdinucleotides.

[0054]FIG. 14: Detection of methylation using dinucleotide synthesis.Dinucleotide synthesis can be used to assess the overall methylationstate of DNA. In the presence of RNA polymerase, CTP or a CTP analog(R₁—C—OH), and GTP or a GTP analog (R₁—CpG—R₂), the deaminatedmethylated DNA template will produce n copies of a labeled dinucleotideproduct, where n is proportional to the number of methylated CpGdinucleotides in the starting DNA. The deaminated unmethylated DNAtemplate can produce no dinucleotide with these substrates because thetemplate no longer encodes “C” at any position.

[0055] If R₁ and R₂ are appropriately labeled, the dinucleotide willproduce a signal that is proportional to the number of methylated CpGsites. For example, if R₁ is a fluorescent energy donor or acceptor thatis compatible with a second donor or acceptor, R₂, a signal will bedetected by fluorescent resonance energy transfer (FRET) between R₁ andR₂ only when the two groups are brought into proximity afterincorporation into the dinucleotide in an enzymatic, template-dependentreaction. The reiterative synthesis of these dinucleotides duringabortive transcription results in multiple signals from each CpG targetand can be used to assess the methylation level of the DNA.

[0056] Similarly, abortive synthesis of trinucleotides by transcriptioninitiation with labeled dinucleotides that end in C (ApC, CpC, GpC, UpC)and termination with labeled GTP can be used to produce signal from thedeaminated methylated template, but not the deaminated unmethylatedtemplate. This trinucleotide synthesis approach may be expanded by theaddition of a site-specific oligonucleotide to allow assessment of themethylation status of a specific CpG site, rather than the entireisland, as illustrated in FIG. 15.

[0057]FIG. 15: Assessing methylation status of specific CpG sites in CpGislands by abortive initiation. Target site probes can be used toexamine the methylation status of specific CpG islands in specificgenes. In the deaminated methylated DNA, the dinucleotide CpG is encodedby the template at the 3 methylated sites 1, 3 and 4, but not by theunmethylated site 2. To specifically determine if Site 3 is methylatedand if so, to what extent, position (C21) can be targeted with a TargetSite Probe, as described in FIG. 11. The template C in question ispositioned at the junction of the bubble region and the downstreamduplex so that it encodes the next incorporated nucleotide forappropriately primed RNA polymerase that binds to the bubble region. Ifa labeled initiator R₁—N_(X)pC—OH is used, where N_(X) may be C for adinucleotide CpC initiator or N_(X) may be CpC for a trinucleotideinitiator, etc., the initiator can be elongated with a labeled GTPanalog pppG—R_(2G) to form a trinucleotide R₁N_(X)CpG—R₂. Similarly, ifthe C in question was not methylated, the position will now be a U andwill encode nucleotide A. If an ATP analog pppA-R_(2A) is present, itwill be incorporated opposite positions where the C was not methylated.If the GTP analog is labeled with group R_(2G), which is an energyacceptor from the R group on the initiator, R₁, then the amount ofR₁N_(x)CpGR_(2G), which will be proportional to the amount of methylatedC present at that position, can be quantified by measuring the emissionfrom R_(2G) at wavelength λ_(2GE). The similar situation exists forincorporation of the ATP analog and measurement of the emission from itsR group, also an energy acceptor from the initiator R₁. By determiningthe ratio of the magnitude of emission from the GTP analog to the totalemission from both the ATP and GTP analogs, the site can be assigned amethylation index M. If all of the Cs at that position are methylated,M=1. If none of the site is methylated, M=0.

[0058]FIG. 16: Genes with altered methylation in cancer. Forty-ninegenes with methylation changes associated with cancer initiation andprogression are plotted versus 13 cancers. An oval indicates an abnormalmethylation status for a gene, coded by cancer type. Cancer is activelyprevented through the expression of close to 100 tumor-suppressor genesthat regulate the cell-division cycle. CpG methylation potentially is apowerful biomarker for cancer detection. Examination of the promoters oftumor suppressor genes from tumor biopsies suggests that CpG methylationis common enough to equal the impact of mutagenesis in tumor promotion.At least 60 tumor suppressor and repair genes are associated withabnormally high levels of CpG methylation across virtually all of thecommon tumor types. In virtually all cases, defective expression oftumor suppressor genes begins at an early stage in tumor progression.Detection of these early methylation events before advanced symptomsappear should improve the chances that a cancer will be treated while itis highly curable. CpG methylation patterns are frequently biased toparticular genes in particular types of cancers. Therefore, it should bepossible to develop methylation signatures for common cancers,indicating both cancer type and stage. Data on the methylation status ofmultiple promoters could give clues as to the location of a tumor incases where several organs can contribute to a sample. For example, shedbladder, kidney or prostate cells can populate a urine sample. Tumorsfrom each of these tissues are frequently associated with distinctcombinations of CpG island methylation.

[0059]FIG. 17: Single nucleotide polymorphism detection by abortiveoligonucleotide synthesis. The identity of a nucleotide at a specificposition can also be determined by abortive initiation in the presenceof target nucleic acid and a position-specific Target Site Probe. Thiscan be applied to SNP identification by initiating transcription with anoligonucleotide complementary to the DNA upstream from the SNP site. Forexample for synthesis of a trinucleotide, the dinucleotide initiatorwould be complementary to the know nucleotides at positions −1 and −2,relative to the SNP site.

[0060]FIG. 18: Detection and identification of single nucleotidepolymorphisms (SNPs) by abortive transcription. The identity of aspecific DNA nucleotide (A,C,G,T/dU) can be identified by abortivetranscription with the use of a Target Site Probe (TSP). For example, todetermine whether a DNA contains a normal nucleotide (wild type) or amutant nucleotide (point-mutation, single nucleotide polymorphism/SNP),a gene-specific TSP can be added to target DNA (oramplification/replication product) such that the SNP positioncorresponds to the last nucleotide in the C/C′ hybrid at the junction ofthe downstream duplex and the bubble region. A labeled initiatoroligonucleotide (R₁NI—OH) that is complementary to the region upstreamof the SNP site can be elongated by an RNA polymerase to add the nextencoded nucleotide, corresponding to the SNP. The labeled terminators(pppN_(T)—R₂ or pppU—R_(2U), pppA—R_(2A), pppC—R_(2C), pppG—R_(2G)) caneach be labeled with different R groups, for example, R_(2A), R_(2C),R_(2G) and R_(2U) could each be resonance energy acceptors from R₁, witheach emitting light with a different detectable wavelength.

[0061]FIG. 19: Signal Generation from abortive promoter. An AbortivePromoter Cassette (APC) consists of one or more oligonucleotides orpolynucleotides that together create a specific binding site for an RNApolymerase coupled to a linker region (APC linker) for attachment totarget molecules (DNA, RNA, Protein). The APC may contain an artificialpromoter, or it may contain the promoter for a specific RNA polymerase.For example, trinucleotide or tetranucleotide products that could begenerated from with a common phage RNA polymerase can be made with alabeled GpA or GpApA initiator and a labeled pppG or pppA terminator.

[0062]FIG. 20: Detection of nucleic acids by abortive transcription. Fordetection of nucleic acids, such as DNA or RNA associated with specificdiseases or with viral and bacterial pathogens, one can either detectthe nucleic acid directly or after replication or primer extension. Inthe first case, the APC linker in the Abortive Promoter Cassette wouldbe designed to be complementary to a known DNA or RNA sequence of thetarget nucleic acid. Alternatively, one or more copies of the target DNAor cDNA copies of target RNA can be made by primer extension or reversetranscription initiated with primers containing a universal APC linkersequence at the 5′ end. In either case, the target DNA or RNA can beretrieved from the sample by attachment to a solid support, for example,to which an oligonucleotide that contains a second target-specificsequence, which is termed a “capture sequence,” has been attached viaany number of immobilization tags, including but not limited to biotin,hexahistidine or any other hapten. Once attached, abortive transcriptionis initiated by addition of a polymerase and the appropriate labelednucleotides, which results in signal generation, as previouslydescribed.

[0063]FIG. 21: Detection of mRNA by Abortive Transcription. An AbortivePromoter Cassette for detection of mRNA will contain as its APC linkeran oligo T tail. This tail is complementary to the poly A tail found atthe 3′ end of eukaryotic mRNAs and will be used for attachment of theAPC to the target mRNA. The target mRNA can be retrieved from a sampleby attachment to an immobilized capture probe containing a capturesequence, which is complementary to some region of the target mRNA.

[0064]FIG. 22: Detection of proteins or other haptens/antigens withabortive transcription. Signal generation via abortive initiation froman Abortive Promoter Cassette can be used to detect other molecules,such as proteins. For example, an APC linker sequence can be prepared towhich thiol-reactive or amine-reactive protein crosslinking agents Rwill be covalently attached. The reactive APC linker will be added tothe target protein, which may be purified or in a complex mixture (suchas a cell lysate), and the APC linker will be covalently attached to thetarget protein via modification of protein thiol and/or amine groups.The labeled protein can then be immobilized utilizing a target-specificprobe (such as an antibody). The Abortive Promoter Cassette is thenattached via the APC linker, and signal is generated, as previouslydescribed.

[0065]FIG. 23: Enhanced detection of molecular targets via abortivetranscription on APC particles. Even greater detection sensitivity canbe achieved with the use of particles to which multiple copies,including tens, hundreds, thousands, tens of thousands or even more ofthe Abortive Promoter Cassette (APC) have been attached. The sphere willalso contain a linker that will be specific for binding to a group thatcan be attached to the target molecule. For example, streptavidin (SA)can be attached to the APC particles and biotin to the target molecule,which can then be immobilized via interaction with a target-specificcapture probe. Once the APC particles interact with the target, forexample via the SA-biotin interaction, polymerase and labelednucleotides can be added for signal generation, as described.

[0066]FIG. 24: Coating of DNA or RNA targets with APC particles forultra-sensitive detection or molecular imaging. An alternative methodfor the ultra-sensitive detection or visualization of target DNA or RNAcan be achieved by reverse transcription of target RNA or copying(single copy or amplification) of target DNA in the presence of probelabeled dNTP analogs. As an example. 5-SH-dUTP can be incorporated atvery high frequency in DNA molecules, which can then be immobilized andfurther modified with other groups, such as biotin. To this, APCparticles can be added, as described in FIG. 23, each of which willinteract with a nucleotide analog on the target. This essentially coatsthe target DNA or RNA with APC particles capable of generating multipleoligonucleotide products for a variety of methods of moleculardetection.

[0067]FIG. 25. Detection of telomerase activity with reiterativeoligonucleotide synthesis. Reiterative oligonucleotide synthesis withDNA polymerases can also be used for signal generation, however, theproduct oligonucleotides need not be released, but may be joinedtandemly in the product. As an example, telomerase activity can bedetected by immobilizing a telomerase-specific probe to a solid matrixto capture cellular telomerase, which carries its own RNA template forDNA synthesis. For example, with human telomerase, the RNA template onthe enzyme encodes the DNA sequence GGGTTA. The capture probe maycontain the sequence GGGTTA, which will be added reiteratively to theend of the telomerase capture probe, if telomerase is present in thesample. Signal generation can be achieved in several ways, one of whichinvolves including one or more reporter tagged dNTPs in the synthesisreaction to produce a product that has multiple R₁ groups attached alongthe backbone of the DNA product. For detection, this product can then behybridized to a complementary probe containing nucleotides with a secondR group (R₂) attached that will hybridize to the R₁ labeled product.This will bring the R₁ and R₂ groups together for signal generation viaFRET from between R₁ and R₂, or via other methods. Alternatively,telomerase may incorporate 2 labeled nucleotides in the product DNA andlook for energy transfer between the 2 labeled nucleotides in the singlestrand of DNA.

[0068]FIG. 26. Synthesis of a dye labeled initiator. 5′EADANS-S-CMP wassynthesized from the conjugation of IAEDANS and (α-S-CMP. The scannedimage of the thin layer chromatography plate shows the control IAEDANSand the IAEDANS labeled product. Lane 1:Cytidine-5′-O-(1-Thiomonophosphate); Lane 2:Cytidine-5′-O-(1-Thiotriphosphate); Lane 3:5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid(1,5-IAEDANS); Lane 4: Cytidine-5′-O-(1-Thiotriphosphate) and(1,5-IAEDANS); Lane 5: Cytidine-5′-O-(1-Thiomonophosphate) and(1,5-IAEDANS); Lane 6: Adenosine-5′-O-(1-Thiomonophosphate); Lane 7:Adenosine-5′-O-(1-Thiomonophosphate) and (1,5-IAEDANS); Lane 8:(1,5-IAEDANS); Lane 9: Cytidine-5′-O-(1-Thiotriphosphate); Lane 10:Cytidine-5′-O-(1-Thiomonophosphate). Lanes 4, 5, and 7 also contain 1 Uof E. coli RNA polymerase, Buffer T and 150 ng of denatured pBR322

[0069]FIG. 27. Abortive Transcription Initiation with labeledinitiators. The photograph of the gel shows the results of an abortivetranscription initiation reaction using three different dinucleotideinitiators, which were (1) ApG; (2) Biotin-ApG; and (3) 5′ TAMRA-SpApG,and a terminating nucleotide, which was α³²P-UTP. All threedinucleotides allowed for incorporation of UTP in the 3^(rd) position togenerate 5′ TAMARA-SpApGpU. The unlabeled ApG incorporates moreefficiently than does the Biotin-ApG, which incorporates moreefficiently than the TAMARA-ApG.

[0070]FIG. 28. Abortive Transcription Initiation with a labeledterminator. The scanned image of the thin layer chromatography plateshows the results of an abortive transcription initiation reaction usingan unlabeled dinucleotide initiator, ApG, and a labeled terminator,which was 5′-SF-UTP (5-thioacetemidofluorescein-uridine 5′-triphosphate.The labeled terminator was efficiently incorporated to generate theoligonucleotide product ApGpU.

[0071]FIG. 29. Abortive transcription initiation reaction with a labeledinitiator and a labeled terminator. The labeled dinucleotide initiator5′TAMARA SpApG was mixed with the labeled terminator, SF-UTP, togenerate the oligonucleotide product, TAMARA-SpApGpU-SF. The formationof TAMARA-SpApGpU-SF was measured in a temperature controlled microtiterplate reader by fluorescence energy transfer. The plate was set to readevery hour at the following parameters: Excitation 485, Emission 620;Gain 35, 99 reads per well per cycle. (A) The signal over background.Background is defined as a well containing only distilled water. Areading was taken every hour for 12 hours starting at time 0.Fluorescein is excited using a 360 nm wavelength filter; the resultingemission peak is at 515 nm. If the TAMRA is in close proximity to thefluorescein it becomes excited as its peak excitation is at 542 nmresulting in an emission peak of 568 nm. (B) The signal over mockreaction. The mock reaction contains all the components of the reactionexcept the E. coli RNA polymerase and the pBR322 plasmid. A reading wastaken every hour for 12 hours starting at time 0. Fluorescein is excitedusing a 360 nm wavelength filter; the resulting emission peak is at 515nm. If the TAMARA is in close proximity to the fluorescein, it becomesexited as its peak excitation is at 542 nm resuling in an emission peakof 568 nm. (C) The signal over SF-UTP. The SF-UTP reaction contains allthe components of the reaction except in place of TAMARA-ApG, itcontains an unlabeled ApG. A reading was taken every hour for 12 hoursstarting at time 0. Fluorescein is excited using a 360 nm wavelengthfilter; the resulting emission peak is at 515 nm. If the TAMARA is inclose proximity to the fluorescein, it becomes exited as its

[0072]FIG. 30. Portion of the contig sequence of the CDKN2A gene. Thesequence represents a small portion of the contig starting at 856630nucleotides from the start of the contig sequence. The sequencerepresents a CpG island. Contig number: NT_(—)008410.4.

[0073]FIG. 31. Schematic representation of a “capture probe” todetermine the methylation status of a specific gene. Oligonucleotideprobes that are specific for a region near the CpG island of the targetgene are immobilized onto a microtiter plate. The DNA of interest isadded to the immobilized probe and bound to the capture probe. The DNAis then chemically modified to convert unmethylated C to T, and leavemethyl-C unaffected. The converted DNA can then be amplified by anoptional PCR step to further enhance the signal. A labeled CpG initiatoris then added with an RNA polymerase and labeled nucleotide(s).

DETAILED DESCRIPTION OF THE INVENTION

[0074] The invention provides methods and kits for detecting thepresence of a target molecule (such as nucleic acid sequence or protein)by generating multiple detectable oligonucleotides through reiterativesynthesis events on a defined nucleic acid. The methods generallycomprise using a labeled nucleotide or oligonucleotide transcriptioninitiator to initiate synthesis of an abortive oligonucleotide productthat is substantially complementary to a defined site on a targetnucleic acid; using a chain terminator to terminate the polymerizationreaction; and, optionally, using either (1) a target site probe to forma transcription bubble complex which comprises double-stranded segmentson either side of a single-stranded target site or (2) an abortivepromoter cassette comprising a transcription bubble region whichincludes a target site or (3) an abortive promoter cassette that isattached to any target molecule and then used to generate a signal.

[0075] In accordance with one aspect, the invention provides methods ofsynthesizing multiple abortive oligonucleotide transcripts from portionsof a target DNA or RNA sequence, wherein the methods comprise combiningand reacting the following: (a) a single-stranded target nucleic acidcomprising at least one target site; (b) an RNA initiator that iscomplementary to a site on the target nucleic acid that is upstream ofthe target site; (c) an RNA polymerase; (d) optionally, nucleotidesand/or nucleotide analogs; (e) a chain terminator; and (f) optionally,either (1) a target site probe that partially hybridizes to a targetregion on the target nucleic acid, forming a transcription bubblecomplex that includes first and second double-stranded regions on eitherside of a single-stranded target site or (2) an abortive promotercassette comprising a transcription bubble region that includes atranscription start site. The combination is subjected to suitableconditions, as described below, such that (a) a target site probehybridizes with a target nucleic acid in a target region that includesthe target site; (b) an RNA initiator hybridizes upstream of a targetsite; (c) an RNA polymerase utilizes the RNA initiator to initiatetranscription at the target site, elongation occurs, and anoligonucleotide transcript is synthesized; (d) a chain terminatorterminates transcription during elongation; (e) the RNA polymerasereleases the short, abortive oligonucleotide transcript withoutsubstantially translocating from the polymerase binding site ordissociating from the template; and (f) steps (c) through (e) arerepeated until sufficient signal is generated and the reaction isstopped. Alternatively, (a) an abortive promoter cassette hybridizeswith an end of the target nucleic acid; (b) an RNA initiator hybridizesupstream of a transcription start site; (c) an RNA polymerase utilizesthe RNA initiator to initiate transcription at the target site,elongation occurs, and an oligonucleotide transcript is synthesized; (d)a chain terminator terminates transcription during elongation; (e) theRNA polymerase releases the short, abortive oligonucleotide transcriptwithout substantially translocating from the polymerase binding site ordissociating from the template; and (f) steps (c) through (e) arerepeated until sufficient signal is generated and the reaction isstopped.

[0076] General Techniques

[0077] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular Biology Techniques Manual,” third edition, (Coyne et al.,2001); “Short Protocols in Molecular Biology,” fourth edition, (Ausubelet al., 1999) “Molecular Cloning: A Laboratory Manual”, second edition(Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods inEnzymology” (Academic Press, Inc.); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR:The Polymerase Chain Reaction” (Mullis et al., eds., 1994).

[0078] Primers, initiators, oligonucleotides, and polynucleotidesemployed as reactants in the present invention can be generated usingstandard techniques known in the art or may be obtained from commercialsources, including but not restricted to Sigma/Aldrich, MolecularProbes, Trilink Technologies.

[0079] Terms

[0080] To facilitate understanding of the invention, the following termshave the following meanings unless expressly stated otherwise:

[0081] “About” as used herein means that a number referred to as “about”comprises the recited number plus or minus 1-10% of that recited number.For example, “about” 50 nucleotides can mean 45-55 nucleotides or as fewas 49-51 nucleotides depending on the situation.

[0082] “Transcription” is an enzyme-mediated process that synthesizes acomplementary RNA transcript that corresponds to a nucleic acid templatesequence. Transcription typically includes three phases, namely,initiation, elongation, and termination. The transcript of the templateis processively synthesized by a polymerase through the formation of aphosphodiester bond between an initiator, which may be a mononucleoside,a mononucleotide, an oligonucleotide, or polynucleotide, and asubsequent NTP, et cetera., without the dissociation of either thenascent transcript or the polymerase from the template, until thepolymerase reaches either a termination sequence on the template or theend of the template sequence or is stopped by other means, such asprotein-DNA transcription roadblocks. As used in typical hybridizationassays, the termination of transcription is generally achieved when thepolymerase completes the elongation phase and reaches the end of thetemplate sequence or a specific transcription termination signal aftertranslocating from the initial enzyme binding site (promoter) on thetemplate. In this context, “translocation” means that the polymerasemoves along the template sequence from an initial enzyme binding site onthe template to another point on the template which is at least 50nucleotides downstream of the enzyme binding site.

[0083] “Abortive transcription” is an enzyme-mediated process thatreiteratively initiates and terminates the synthesis of oligonucleotidesthat correspond to at least one portion, or target site, of acomplementary nucleic acid template sequence. The abortiveoligonucleotides synthesized vary in length of nucleotides, and maycontain from about 2 to about 26 nucleotides, about 26 to about 50nucleotides and about 50 nucleotides to about 100 nucleotides, andgreater than 100 nucleotides.

[0084] “Abortive transcription” also includes three phases, namely,initiation, elongation, and termination. During the initiation phase, apolymerase forms a phosphodiester bond between an initiator and a secondNTP, and then adds subsequent NTPs, et cetera., transcribing thetemplate sequence to synthesize an oligonucleotide transcript of fromabout 2 to about 50 nucleotides in length and then terminating thetranscription event by releasing the nascent oligonucleotide transcript,without the polymerase substantially translocating from the polymerasebinding site or dissociating from the template. In other words, the RNApolymerase substantially remains at the initial binding site on thetemplate, releases a first nascent oligonucleotide transcript, and thenis capable of engaging in another transcription initiation event toproduce a second oligonucleotide transcript, which is substantiallycomplementary to substantially the same target site that was transcribedto produce the first oligonucleotide transcript. In this manner, thepolymerase reiteratively transcribes a single portion of the template(i.e., a target region) and releases multiple copies of substantiallyidentical nascent oligonucleotide transcripts.

[0085] “Reverse transcription” refers to the transcription of an RNAtemplate to synthesize complementary DNA (cDNA).

[0086] “Reiterative” refers to multiple identical or highly similarcopies of a sequence of interest.

[0087] “Replication” is an enzyme-mediated process which synthesizes acomplementary nucleic acid molecule from a single-stranded nucleic acidtemplate sequence. The DNA replicate of the template is synthesized by aDNA polymerase through the formation of a phosphodiester bond between aprimer and a first deoxyribonucleoside triphosphate (dNTP), followed bythe formation of a second phosphodiester bond between the first dNTP anda subsequent dNTP, et cetera., without the dissociation of either theDNA replicate or the DNA polymerase from the template, until the DNApolymerase reaches either a termination sequence on the template or theend of the template sequence. In a typical DNA primer extensionreaction, replication of the template terminates when the DNA polymerasesynthesizes the entire template sequence after translocating from theinitial enzyme binding site on the template. In this context,“translocation” means that the DNA polymerase moves along the templatesequence from an initial enzyme binding site on the template to anotherpoint on the template which is downstream of the enzyme binding site.

[0088] “Oligonucleotide product” refers to the oligonucleotide that issynthesized by the reiterative synthesis reaction of the presentinvention. An oligonucleotide product may be an “oligonucleotidetranscript,” if the polymerization reaction is a transcription reactioncatalyzed by an RNA polymerase, or an “oligonucleotide repeat,” if thepolymerization reaction is a DNA synthesis reaction catalyzed bytelomerase or DNA polymerase.

[0089] “Termination” refers to the use of a chain terminator to concludea chain elongation or primer extension reaction that is catalyzed by apolymerase. A “chain terminator” or “terminator” may comprise anycompound, composition, complex, reactant, reaction condition, or processstep (including withholding a compound, reactant, or reaction condition)which inhibits the continuation of transcription by the polymerasebeyond the initiation and/or elongation phases. A “chain terminatingnucleotide” is a chain terminator that comprises a nucleotide ornucleotide analog that inhibits further chain elongation onceincorporated, due to either the structure of the nucleotide analog orthe sequence of the nucleic acid being copied or transcribed.

[0090] A “target sequence” or “target polynucleotide” is apolynucleotide sequence of interest for which detection,characterization or quantification is desired. The actual nucleotidesequence of the target sequence may be known or not known.

[0091] A “target site” is that portion of the target sequence that isdetected by transcription by a polymerase to form an oligonucleotideproduct. In accordance with the invention, there is at least one targetsite on a target nucleic acid. The sequence of a target site may or maynot be known with particularity. That is, while the actual geneticsequence of the target nucleic acid may be known, the genetic sequenceof a particular target site that is transcribed or replicated by apolymerase need not be known.

[0092] A “target region” is that portion of a target sequence to which atarget site probe partially hybridizes to form a bubble complex, asdescribed in detail below. In accordance with the invention, there is atleast one target region on a target nucleic acid, and each target regioncomprises a target site. The sequence of a target region is known withsufficient particularity to permit sufficiently stringent hybridizationof a complementary target site probe, such that the target site probeforms a bubble complex with the target region.

[0093] Generally, a “template” is a polynucleotide that contains thetarget nucleotide sequence. In some instances, the terms “targetsequence”, “template polynucleotide”, “target nucleic acid”, “targetpolynucleotide”, “nucleic acid template”, “template sequence”, andvariations thereof, are used interchangeably. Specifically, the term“template” refers to a strand of nucleic acid on which a complementarycopy is synthesized from nucleotides or nucleotide analogs through theactivity of a template-dependent nucleic acid polymerase. Within aduplex, the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand. The “template” strand mayalso be referred to as the “sense” strand, and the non-template strandas the “antisense” strand.

[0094] “Synthesis” generally refers to the process of producing at leastone complementary copy of a target site or other portion of a targetsequence. “Multiple copies” means at least 2 copies. A “copy” does notnecessarily mean perfect sequence complementarity or identity with thetemplate sequence. For example, copies can include nucleotide analogs,intentional sequence alterations (such as sequence alterationsintroduced through a primer comprising a sequence that is hybridizable,but not complementary, to the template), and/or sequence errors thatoccur during synthesis. “Synthesis” encompasses both transcription of atarget nucleic acid and replication of a target nucleic acid.

[0095] “Polynucleotide” or “nucleic acid strand”, as usedinterchangeably herein, refers to nucleotide polymers of any length,such as two or more, and includes both DNA and RNA. The nucleotides canbe deoxyribonucleotides, ribonucleotides, nucleotide analogs (includingmodified phosphate moieties, bases, or sugars), or any substrate thatcan be incorporated into a polymer by a suitable enzyme, such as a DNApolymerase or an RNA polymerase. Thus, a polynucleotide may comprisemodified nucleotides, such as methylated nucleotides, and their analogs.If present, modification to the nucleotide structure may be impartedbefore or after synthesis of the polymer. The sequence of nucleotidesmay be interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications include, for example,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, cabamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,glutathione-s-transferase, methylases, demethylases, DNA repair enzymes,nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.),those with intercalators (e.g., ethidium, acridine, psoralen, etc.),those with antibody-specific haptens (dinitrophenyl (DNP), biotin,etc.), those with affinity tags (hexahistadine, glutathione, etc.),those containing chelators (e.g., metals, radioactive metals, boron,oxidative metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.), those withchemical or photochemical activities (DNA or RNA cleavage agents,crosslinkers, fluorescent compounds, etc.) as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent on the pentose (i.e., ribose or deoxyribose) ring of anucleotide may be, for example, replaced by phosphonate or phosphategroups, protected by standard protecting groups, activated to prepareadditional linkages to additional nucleotides, or conjugated to a solidsupport. The 5′ and 3′ terminal OH groups on the pentose ring of anucleotide can be phosphorylated or substituted with amines or organiccapping group moieties of from about 1 to about 50 carbon atoms. Otherhydroxyl groups on the ribose or deoxyribose ring may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example,2′-O-methyl-2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugaranalogs, anomeric sugars, epimeric sugars, such as arabinose, xylose,pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, andabasic nucleoside analogs such as methyl riboside. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

[0096] “Nucleotide” or “NTP” refers to a base-sugar-phosphate compound.“Base” refers to a nitrogen-containing ring molecule that, when combinedwith a pentose sugar and a phosphate group, form a nucleotide. Basesinclude single ring pyrimidines, such as cytosine (C), thymine (T), anduracil (U), and double ring purines, such as adenine (A) and guanine(G). “Sugar” or “pentose sugar” generally refers to a pentose ring, suchas a ribose ring or deoxyribose ring. Nucleotides are the monomericsubunits of both types of nucleic acid polymers, that is, RNA and DNA.“Nucleotide” or “NTP” refers to any nucleoside 5′ phosphate, that is,ribonucleoside 5′ phosphates (i.e., mono-, di-, and triphosphates) anddeoxyribonucleoside 5′ phosphates (i.e., mono-, di-, and triphosphates),and includes “nucleoside phosphate analogs”, “nucleotide analogs”, and“NTP analogs”. “Nucleoside phosphate analog”, “nucleotide analog”, and“NTP analog” refer to any nucleoside 5′ phosphate (i.e., mono-, di-, ortriphosphate) which is analogous to a native nucleotide but whichcontains one or more chemical modifications when compared to thecorresponding native nucleotide. Nucleotide analogs includebase-modified analogs (e.g. 5-mercapto pyrimidines, 8-mercapto purines),phosphate-modified analogs (e.g., α-thio-triphosphates), andsugar-modified analogs (3′ OMe, 3′deoxy) and may comprise modified formsof deoxyribonucleotides as well as ribonucleotides.

[0097] “Nucleoside” refers to a base-sugar combination without aphosphate group. Nucleosides include, but are note limited to, adenosine(A), cytidine (C), guanosine (G), thymidine (T), and uridine (U).

[0098] The term “oligonucleotide” generally refers to short, typicallysingle-stranded, synthetic polynucleotides that are generally, but notnecessarily, less than about 200 nucleotides in length. Moreparticularly, an oligonucleotide may be defined as a molecule comprisedof two or more nucleotides, including deoxyribonucleotides and/orribonucleotides. The exact size depends on many factors, which in turndepend on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, degradation of longer DNA or RNA,transcription, reverse transcription, abortive transcription orreiterative synthesis, as further described herein, and a combinationthereof.

[0099] Because mononucleotides undergo a reaction which synthesizesoligonucleotides by covalently bonding the 3′ oxygen of a firstmononucleotide pentose ring to the 5′ phosphate of a secondmononucleotide through a phosphodiester linkage, a first end of anoligonucleotide is referred to as the “5′ end” if the 5′ phosphate ofthe terminal nucleotide is not linked to a 3′ oxygen of a nucleotidepentose ring, and a second end of an oligonucleotide is referred to asthe “3′ end” if the 3′ oxygen of the terminal nucleotide is not linkedto a 5′ phosphate of a subsequent nucleotide pentose ring. As usedherein, a nucleic acid sequence, even if the sequence is internal to alarger oligonucleotide, also may be said to have 5′ and 3′ ends. Forsingle-stranded DNA or RNA, a first region along a nucleic acid strandis said to be “upstream” of a second region, if the 3′ end of the firstregion is before the 5′ end of the second region when moving along astrand of nucleic acid in a 5′→3′ direction. Conversely, a first regionalong a nucleic acid strand is said to be “downstream” of a secondregion, if the 5′ end of the first region is after the 3′ end of thesecond region when moving along a strand of nucleic acid in a 5′→3′direction.

[0100] The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide that is 3′ (downstream) from anotherregion or position in the same polynucleotide or oligonucleotide whenmoving along the polynucleotide or oligonucleotide in a 5′→3′ direction.

[0101] The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide that is 5′ (upstream) from anotherregion or position in the same polynucleotide or oligonucleotide whenmoving along the polynucleotide or oligonucleotide in a 5′→3′ direction.

[0102] “Nucleic acid sequence” refers to an oligonucleotide orpolynucleotide, and fragments, segments, or portions thereof, and to DNAor RNA of genomic or synthetic origin, which may be single- ordouble-stranded, and represents either the sense or the antisensestrand.

[0103] The term “substantially single-stranded”, when used in referenceto a nucleic acid substrate, means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two substantiallycomplementary segments or regions of nucleic acid that are held togetherby inter-strand or intra-strand base pairing interactions.

[0104] As used herein, the terms “complementary” or “complementarity”are used in reference to a first polynucleotide (which may be anoligonucleotide) which is in “antiparallel association” with a secondpolynucleotide (which also may be an oligonucleotide). As used herein,the term “antiparallel association” refers to the alignment of twopolynucleotides such that individual nucleotides or bases of the twoassociated polynucleotides are paired substantially in accordance withWatson-Crick base-pairing rules. For example, the sequence “A-G-T” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the polynucleotides' bases are matched accordingto the base pairing rules. Or, there may be “complete” or “total”complementarity between the polynucleotides. The degree ofcomplementarity between the polynucleotides has significant effects onthe efficiency and strength of the hybridization between twopolynucleotides. This is of particular importance in synthesisreactions, as well as detection methods which depend upon bindingbetween polynucleotides. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically by considering anumber of variables, including, for example, the length of the firstpolynucleotide, which may be an oligonucleotide, the base compositionand sequence of the first polynucleotide, and the ionic strength andincidence of mismatched base pairs. A general formula that may be usedto calcuate the melting temperature of an oligonucleotide is:Tm=(2(UA)+4(GC))−0.5 C for every 1% formamide. For DNA-DNA hybrids, theTm is approximated by the following formula: Tm=81.5+16.6 (log M)+0.41(%G+C)−500/L; M is the molarity of the monovalent cations; L is thelength of the hybrid base pairs (Anal Biochem. 138:267-284, 1984).

[0105] The terms “self-complementary” and “self-complementarity”, whenused in reference to a polynucleotide (e.g., an oligonucleotide), meanthat separate regions of the polynucleotide can base-pair with eachother. Because this term refers only to intramolecular base-pairing, anystrand said to have a region of self-complementarity must have at leasttwo regions capable of base-pairing with one another. As defined above,complementarity may be either “complete” or “partial”. As used inreference to the oligonucleotides of the present invention, regions ofan oligonucleotide are considered to have significantself-complementarity when these regions are capable of forming a duplexof at least 3 contiguous base pairs (i e., three base pairs of completecomplementarity), or when they may form a longer duplex that ispartially complementary.

[0106] The term “primer” generally refers to a short, single-strandedoligonucleotide which has a free 3′-OH group and which can bind to andhybridize with a target sequence that is potentially present in a sampleof interest. After hybridizing to a target sequence, a primer is capableof promoting or initiating polymerization or synthesis of apolynucleotide or oligonucleotide extension product that iscomplementary to the target sequence or a portion of the targetsequence. A primer is selected to be “substantially” complementary to aspecific portion of a target nucleic acid sequence. A primer issufficiently complementary to hybridize with a target sequence andfacilitate either transcription or replication of a portion of thetarget nucleic acid. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the templatestrand. Non-complementary bases can be interspersed within the primer,provided that the primer sequence has sufficient complementarity withthe template sequence to hybridize with the template and thereby form atemplate-primer complex for initiating synthesis of a polynucleotide oroligonucleotide product.

[0107] The term “initiator” refers to a mononucleoside, mononucleotide,oligonucleotide, polynucleotide or analog thereof, which is incorporatedinto the 5′ end of a nascent RNA molecule and may be considered a“primer” for RNA synthesis (“initiator primer”).

[0108] In one embodiment, an RNA initiator facilitates the initiation oftranscription at a target site on a single-stranded target nucleic acidin the absence of a template promoter sequence, as is known in the art.(See, U.S. Pat. No. 5,571,669; Daube and von Hippel, Science, 258:1320-1324 (1992)). In another embodiment, initiators are used torandomly start abortive transcription at a plurality of target sites onthe nucleic acid template (FIG. 16). The initiators and/or theindividual nucleotides or nucleotide analogs that are used to extend theinitiators may be suitably modified to enable signal generation,detection of the oligonucleotide products, and a determination of thepresence or absence of the target sequence.

[0109] For example, it may be desirable to modify the initiator toprovide the initiator with a label moiety for a variety of purposes,including detection of the abortive oligonucleotide product(s). Examplesof such modifications include, but are not limited to, fluorescentmolecules and energy transfer dyes (such as, fluorescein, aedans,coumarine, bodipy dyes, and rhodamine based dyes), fluorescent quenchermolecules (for example, Dabcyl), proteins, peptides, amino linkers, oramino acid based molecules (for example polyhistidine), modified basesand modified and unmodified base analogs, peptide nucleic acids (PNAs),methylphosphonates, radioactive labels, terminal phosphates, 3′glyceryl, other carbohydrate based molecules, fatty acid derivedmolecules, carbon spacer molecules, electrochemiluminescent labels,lanthanide labels, avidin and its derivatives (for example,streptavidin, Neutravidin, etc.), biotin, steroid molecules (such asDigoxygenin), thiol linkages, ferritin labels, and the like.

[0110] As used herein, the term “hybridization” is used in reference tothe base-pairing of complementary nucleic acids, includingpolynucleotides and oligonucleotides. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementarybetween the nucleic acids, the stringency of the reaction conditionsinvolved, the melting temperature (T_(m)) of the formed hybrid, and theG:C ratio within the duplex nucleic acid. Generally, “hybridization”methods involve annealing a complementary polynucleotide to a targetnucleic acid (i.e., the sequence to be detected either by direct orindirect means). The ability of two polynucleotides and/oroligonucleotides containing complementary sequences to locate each otherand anneal to one another through base pairing interactions is awell-recognized phenomenon.

[0111] With regard to complementarity, it may be important for somediagnostic applications to determine whether the hybridization of twopolynucleotides and/or oligonucleotides represents complete or partialcomplementarity. For example, where it is desired to detect simply thepresence or absence of pathogen DNA (such as from a virus, bacterium,fungi, mycoplasma, or protozoan for example), the hybridization methodneed only ensure that hybridization occurs when the relevant sequence ispresent; conditions can be selected where both partially complementaryprobes and completely complementary probes will hybridize. Otherdiagnostic applications, however, may require that the hybridizationmethod be capable of distinguishing between partial and completecomplementarity, such as in cases where it may be of interest to detecta genetic polymorphism, that is, a difference in a single base pairbetween multiple alleles (variations) that may exist for a particulargene or genetic marker.

[0112] “Stringency” generally refers to the conditions under whichnucleic acid hybridizations are conducted, including temperature, ionicstrength, and the presence of other compounds. Conditions of “highstringency” generally refer to those conditions under which nucleic acidbase pairing will occur only between polynucleotide and/oroligonucleotide regions that have a high frequency of complementary basesequences. Consequently, conditions of “weak” or “low” stringency may bepreferred when it is desirable to hybridize or anneal twopolynucleotides and/or oligonucleotides, which are not completelycomplementary to one another.

[0113] The term “reactant” is used in its broadest sense. A reactant cancomprise an enzymatic reactant, a chemical reactant, or ultravioletlight (ultraviolet light, particularly short wavelength ultravioletlight, is known to break polynucleotide polymers). Any agent capable ofreacting with an oligonucleotide or polynucleotide to modify theoligonucleotide or polynucleotide is encompassed by the term “reactant,”including a “reactant nucleotide” that is added to a reaction mixturefor incorporation into an oligonucleotide product by a polymerase.

[0114] A “complex” is an assembly of components. A complex may or maynot be stable and may be directly or indirectly detected. For example,as described herein, given certain components of a reaction and the typeof product(s) of the reaction, the existence of a complex can beinferred. For the purposes of this invention, a complex is generally anintermediate with respect to a final reiterative synthesis product, suchas a final abortive transcription or replication product for example.

[0115] A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

[0116] The term “enzyme binding site” refers to a polynucleotide regionthat is characterized by a sequence or structure that is capable ofbinding to a particular enzyme or class of enzymes, such as apolymerase.

[0117] “Polymerase” refers to any agent capable of facilitating orcatalyzing the polymerization (joining) of nucleotides and/or nucleotideanalogs. Suitable agents include naturally occurring enzymes, such asnaturally occurring RNA polymerases (including RNA-dependent andDNA-dependent RNA polymerases), DNA polymerases (including DNA-dependentand RNA-dependent DNA polymerases), as well as modified or mutantenzymes that may currently exist (such as the mutant RNA polymerasesdisclosed in Sousa, et al., U.S. Pat. No. 6,107,037 for example) or maybe hereafter created or designed, which modified or mutant enzymes maybe designed to exhibit characteristics that are desirable for particularapplications. Exemplary characteristics of a modified or mutant enzymemay include, but are not limited to, relaxed template specificity,relaxed substrate specificity, increased thermostability, and/or thelike. It is intended that the term “polymerase” encompasses boththermostable and thermolabile enzymes.

[0118] The term “thermostable” when used in reference to an enzyme, suchas an RNA or DNA polymerase for example, indicates that the enzyme isfunctional or active (i.e., can perform catalysis) at an elevatedtemperature, that is, at about 55° C. or higher. Thus, a thermostablepolymerase can perform catalysis over a broad range of temperatures,including temperatures both above and below about 55° C.

[0119] The term “template-dependent polymerase” refers to a nucleic acidpolymerase that synthesizes a polynucleotide or oligonucleotide productby copying or transcribing a template nucleic acid, as described above,and which does not synthesize a polynucleotide in the absence of atemplate. This is in contrast to the activity of a template-independentnucleic acid polymerase, such as terminal deoxynucleotidyl transferaseor poly-A polymerase for example, that may synthesize or extend nucleicacids in the absence of a template.

[0120] A “DNA-dependent RNA polymerase” is an enzyme which facilitatesor catalyzes the polymerization of RNA from a complementary DNAtemplate.

[0121] A “DNA-dependent DNA polymerase” is an enzyme which facilitatesor catalyzes DNA replication or synthesis, that is, the polymerizationof DNA from a complementary DNA template.

[0122] An “RNA-dependent RNA polymerase” is an enzyme which facilitatesor catalyzes the polymerization of RNA from a complementary RNAtemplate.

[0123] An “RNA-dependent DNA polymerase” or “reverse transcriptase” isan enzyme that facilitates or catalyzes the polymerization of DNA from acomplementary RNA template.

[0124] “Primer extension”, “extension”, “elongation”, and “extensionreaction” is the sequential addition of nucleotides to the 3′ hydroxylend of a mononucleotide, oligonucleotide, or polynucleotide initiator orprimer which has been annealed or hybridized to a longer, templatepolynucleotide, wherein the addition is directed by the nucleic acidsequence of the template and/or the binding position of the polymerase.Extension generally is facilitated by an enzyme capable of synthesizinga polynucleotide or oligonucleotide product from a primer or initiator,nucleotides and a template. Suitable enzymes for these purposes include,but are not limited to, any of the polymerases described above.

[0125] “Incorporation” refers to becoming a part of a nucleic acidpolymer. There is a known flexibility in the terminology regardingincorporation of nucleic acid precursors. For example, the nucleotidedGTP is a deoxyribonucleoside triphosphate. Upon incorporation into DNA,dGTP becomes dGMP, that is, a deoxyguanosine monophosphate moiety.Although DNA does not include dGTP molecules, one may say that oneincorporates dGTP into DNA.

[0126] The terms “sample” and “test sample” are used in their broadestsense. For example, a “sample” or “test sample” is meant to include aspecimen or culture (e.g., microbiological cultures) as well as bothbiological and environmental samples. Samples of nucleic acid used inthe methods of the invention may be aqueous solutions of nucleic acidderived from a biological or environmental sample and separated, bymethods known in the art, from other materials, such as proteins,lipids, and the like, that may be present in the sample and that mayinterfere with the methods of the invention or significantly increasethe “background” signal in carrying out the methods.

[0127] A biological sample may comprise any substance which may includenucleic acid, such as animal (including human) tissue, animal fluids(such as blood, saliva, mucusal secretions, semen, urine, sera, cerebralor spinal fluid, pleural fluid, lymph, sputum, fluid from breast lavage,and the like), animal solids (e.g., stool), cultures of microorganisms,liquid and solid food and feedproducts, waste, cosmetics, or water thatmay be contaminated with a microorganism, or the like. An environmentalsample may include environmental material, such as surface matter, soil,water, and industrial samples, as well as samples obtained from food anddairy processing instruments, apparatus, equipment, utensils, anddisposable and non-disposable items. These examples are merelyillustrative and are not intended to limit the sample types applicableto the present invention.

[0128] “Purified” or “substantially purified” refers to nucleic acidsthat are removed from their natural environment, isolated or separated,and are at least 60% free, preferably 75% free, and most preferably 90%free from other components with which they are naturally associated. An“isolated polynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

[0129] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence, so long as thedesired functional activity is retained.

[0130] A “deletion” is defined as a change in a nucleic acid sequence inwhich one or more nucleotides are absent as compared to a standardnucleic acid sequence.

[0131] An “insertion” or “addition” is a change in a nucleic acidsequence which has resulted in the addition of one or more nucleotidesas compared to a standard nucleic acid sequence.

[0132] A “substitution” results from the replacement of one or morenucleotides in a nucleic acid by different nucleotides.

[0133] An “alteration” in a nucleic acid sequence refers to any changein a nucleic acid sequence or structure, including, but not limited to adeletion, an addition, an addition-deletion, a substitution, aninsertion, a reversion, a transversion, a point mutation, or amicrosatellite alteration, or methylation.

[0134] “Methylation” refers to the addition of a methyl group (—CH₃) toa nucleotide base in DNA or RNA.

[0135] Sequence “mutation” refers to any sequence alteration in asequence of interest in comparison to a reference sequence. A referencesequence can be a wild type sequence or a sequence to which one wishesto compare a sequence of interest. A sequence mutation includes singlenucleotide changes, or alterations of more than one nucleotide in asequence, due to mechanisms such as substitution, deletion, orinsertion. A single nucleotide polymorphism (SNP) is also a sequencemutation as used herein.

[0136] “Microarray” and “array,” as used interchangeably herein, referto an arrangement of a collection of polynucleotide sequences in acentralized location. Arrays can be on a solid substrate, such as aglass slide, or on a semi-solid substrate, such as nitrocellulosemembrane. The polynucleotide sequences can be DNA, RNA, or anycombinations thereof.

[0137] The term “label” refers to any atom, molecule, or moiety whichcan be used to provide a detectable (preferably quantifiable) signal,either directly or indirectly, and which can be attached to anucleotide, nucleotide analog, nucleoside mono-, di-, or triphosphate,nucleoside mono-, di-, or triphosphate analog, polynucleotide, oroligonucleotide. Labels may provide signals that are detectable byfluorescence, radioactivity, chemiluminescence, electrical,paramagnetism, colorimetry, gravimetry, X-ray diffraction or absorption,magnetism, enzymatic activity, and the like. A label may be a chargedmoiety (positive or negative charge) or, alternatively, may be chargeneutral.

[0138] “Detection” includes any means of detecting, including direct andindirect detection. For example, “detectably fewer” products may beobserved directly or indirectly, and the term indicates any reduction inthe number of products (including no products). Similarly, “detectablymore” products means any increase, whether observed directly orindirectly.

[0139] As used herein, the terms “comprises,” “comprising”, “includes”,and “including”, or any other variations thereof, are intended to covera non-exclusive inclusion, such that a process, method, composition,reaction mixture, kit, or apparatus that comprises a list of elementsdoes not include only those elements but may include other elements notexpressly listed or inherent to such process, method, composition,reaction mixture, kit, or apparatus.

[0140] “A,” “an,” “the,” and the like, unless otherwise indicated,include plural forms.

[0141] Components and Reaction Conditions

[0142] Target Nucleic Acid

[0143] The target nucleic acid can be either a naturally occurring orsynthetic polynucleotide segment, and it can be obtained or synthesizedby techniques that are well-known in the art. A target sequence to bedetected in a test sample may be present initially as a discretemolecule, so that the sequence to be detected constitutes the entirenucleic acid, or may be present as only one component of a largermolecule. The target nucleic acid can be only a minor fraction of acomplex mixture, such as a biological sample, and can be obtained fromvarious biological materials by procedures that are well-known in theart. The target nucleic acid to be detected may include nucleic acidsfrom any source, in purified, or unpurified form, which can be DNA(including double-stranded (ds) DNA and single-stranded (ss) DNA) or RNA(including tRNA, mRNA, rRNA), mitochondrial DNA or RNA, chloroplast DNAor RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, orplasmids; and the genomes of biological material, such as the genomes ofmicroorganisms (including bacteria, yeast, viruses, viroids, molds, andfungi), plants, animals, humans, or fragments thereof. Standardtechniques in the art are used to obtain and purify the nucleic acidsfrom a test sample. Methods for the extraction and/or purification ofsuch nucleic acids have been described, for example, by Sambrook, etal., Molecular Cloning: A Laboratory Manual (New York, Cold SpringHarbor Laboratory, third edition, 2000). Detection of an RNA target mayor may not require initial complementary DNA (cDNA) synthesis, as knownin the art. Detection of a DNA-RNA hybrid may require denaturation ofthe hybrid to obtain a ssDNA or denaturation followed by reversetranscription to obtain a cDNA.

[0144] Target Proteins

[0145] In another embodiment of the invention, the target may be anothermolecule, such as a protein, which is labeled by covalent or noncovalentattachment of a defined nucleic acid sequence which can be used forreiterative oligonucleotide synthesis (FIG. 23). The target protein canbe either a naturally occurring or synthetic polypeptide segment, and itcan be obtained or synthesized by techniques that are well-known in theart. A target protein to be detected in a test sample may be presentinitially as a discrete molecule, so that the protein to be detectedconstitutes the entire protein, or may be present as only one componentof a larger complex. The target protein can be only a minor fraction ofa complex mixture, such as a biological sample, and can be obtained fromvarious biological materials by procedures that are well-known in theart. The target protein to be detected may include proteins from anysource, in purified or unpurified form. Standard techniques in the artare used to obtain and purify the proteins from a test sample. Methodsfor the extraction and/or purification of such proteins have beendescribed, for example, by Sambrook, et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory, thirdedition, 2000).

[0146] Immobilization

[0147] In one embodiment of the invention, the target molecule may beimmobilized. In another embodiment, the target molecule may beimmobilized to form, for example, a microarray. A single molecule arrayin accordance with this embodiment includes a solid matrix, abioreactive or bioadhesive layer, and a bioresistant layer. Solid phasesthat are useful as a matrix for the present invention include, but arenot limited to, polystyrene, polyethylene, polypropylene, polycarbonate,or any solid plastic material in the shape of test tubes, beadsmicroparticles, dip-sticks, or the like. Additionally, matrices include,but are not limited to, membranes, microtiter plates (e.g., 96-well and384-well), test tubes, and Eppendorf tubes. Solid phases also includeglass beads, glass test tubes, and any other appropriate shape that ismade of glass. A functionalized solid phase, such as plastic or glass,which has been modified so that the surface carries carboxyl, amino,hydrazide, or aldehyde groups can also be used. In general, suitablesolid matrices comprise any surface to which a bioadhesive layer, suchas a ligand-binding agent, can be attached or any surface which itselfprovides a ligand attachment site.

[0148] The bioadhesive layer can be an ionic adsorbent material such asgold, nickel, or copper (Montemagno and Bachand, ConstructingNanomechanical Devices Powered by Biomolecular Motors, Nanotechnology,10: 225-231 (1999)), protein-adsorbing plastics, such as polystyrene(U.S. Pat. No. 5,858,801), or a covalent reactant, such as a thiolgroup. To create a patterned array in the bioadhesive layer, anelectron-sensitive polymer, such as polymethyl methacrylate (PMMA) forexample, can be used to coat the solid support and can be etched in anydesired pattern with an electron beam followed by development to removethe sensitized polymer. The etched portions of the polymer are thencoated with a metal, such as nickel, and the polymer is removed with asolvent, leaving a pattern of metal posts on the substrate. This methodof electron beam lithography provides the high spatial resolution andsmall feature size which facilitates the immobilization of a singlemolecule at each point in the patterned array. An alternate means forcreating high-resolution patterned arrays is atomic force microscopy. Afurther means is X-ray lithography.

[0149] Antibody or oligonucleotide capture probes can be attached to thebioadhesive pattern by providing a polyhistidine tag on the captureprobe that binds to the metal bioadhesive patterns. The capture probesmay be, for example, from about 15 to about 500 nucleotides in length.Other conventional means for attachment employ homobifunctional andheterobifunctional crosslinking reagents. Homobifunctional reagentscarry two identical functional groups, whereas heterobifunctionalreagents contain two dissimilar functional groups to link the captureprobes to the bioadhesive. The heterobifunctional cross-linking agentsmay contain a primary amine-reactive group and a thiol-reactive group.Covalent crosslinking agents are selected from reagents capable offorming disulfide (S—S), glycol (—CH(OH)—CH(OH)—), azo (—N═N—), sulfone(—S(═O₂—), ester (—C(═O)—O—), or amide (—C(═O)—N—) bridges. Crosslinkingagents include, but are not limited to, maleamides, iodoacetamides, anddisulfies. Table 1 provides a list of representative classes ofcrosslinking reagents and their group specificity (Wong, S. S. Chemistryof Protein Conjugation and Cross-Linking, 1991, CRC Press, Inc., BocaRaton, USA) TABLE 1 Crosslinking Reagents and group specificity ReagentGroup specificity alpha-haloacetyl compounds eg SH, S—CH3, NH2,phenolic, ICH2COOH imadazole N-maleimides SH, NH2 mercurials SHDisulfides SH Aryl halides SH, NH2, phenolic, imidazole Acid anhydrideseg. Succinic NH2, phenolic anhydride Isocyanates eg. HNCO NH2Isothiocyanates R—NCS NH2 Sulfonyl halides NH2 Imidoesters Nh2Diazoacetates COOH, SH Diazonium salts eg phenolic, imidazolebenzene-N2 + Cl- dicarbonyl compound NH—C(NH)—NH2

[0150] A bioresistant layer may be placed or superimposed upon thebioadhesive layer either before or after attachment of the capture probeto the bioadhesive layer. The bioresistant layer is any material thatdoes not bind the capture probe. Non-limiting examples include bovineserum albumin, gelatin, lysozyme, octoxynol, polysorbate 20(polyethenesorbitan monolaurate), and polyethylene oxide containingblock copolymers and surfactants (U.S. Pat. No. 5,858,801). Depositionof the bioadhesive and bioresistant layers may be accomplished byconventional means, including spraying, immersion, and evaporativedeposition (metals).

[0151] In one embodiment, the solid matrix may be housed in a flowchamber having an inlet and outlet to accommodate the multiple solutionsand reactants that are allowed to flow past the immobilized captureprobes. The flow chamber can be made of plastic or glass and may beeither open or transparent in the plane viewed by a microscope oroptical reader. Electro-osmotic flow includes a fixed charge on thesolid support and a voltage gradient (current) passing between twoelectrodes placed at opposing ends of the solid support.

[0152] Primers

[0153] In accordance with the invention, a primer is used to initiatereplication by a DNA polymerase of a target site on the target nucleicacid. If the polymerase is a DNA polymerase, the primer may be comprisedof ribonucleotides or deoxyribonucleotides. The primers and/or theindividual nucleotides or nucleotide analogs that are used to extend theprimers may be suitably modified to enable signal generation, detectionof the oligonucleotide products, and a determination of the presence orabsence of the target sequence.

[0154] The primers used in the practice of the invention may be madesynthetically, using conventional chemical or enzymatic nucleic acidsynthesis technology. In one embodiment, the primers are less than about25 nucleotides in length, usually from about 1 to about 10 nucleotidesin length, and preferably about 2 to 3 nucleotides in length. It may bedesirable to modify the nucleotides or phosphodiester linkages in one ormore positions of the primer. Examples of such modifications include,but are not limited to, fluorescent molecules and energy transfer dyes(such as, fluorescein, aedans, coumarine, bodipy dyes, and rhodaminebased dyes), fluorescent quencher molecules (for example, Dabcyl),proteins, peptides, amino linkers, or amino acid based molecules (forexample polyhistidine), modified bases and modified and unmodified baseanalogs, peptide nucleic acids (PNAs), methylphosphonates, radioactivelabels, terminal phosphates, 3′ glyceryl, other carbohydrate basedmolecules, fatty acid derived molecules, carbon spacer molecules,electrochemiluminescent labels, lanthanide labels, avidin and itsderivatives (for example, streptavidin, Neutravidin, etc.), biotin,steroid molecules (such as Digoxygenin), thiol linkages, ferritinlabels, and the like.

[0155] Target Site Probes

[0156] In accordance with the invention, an oligonucleotide target siteprobe is used to direct a polymerase to a target site on the targetnucleic acid by forming a bubble complex in a target region of thetarget nucleic acid (FIG. 1). The target site probe may vary in thelength of nucleotides, including but not limited to, about 20 to about50 nucleotides, about 51 to about 75 nucleotides, about 76 to about 100nucleotides, and greater than 100 nucleotides. The bubble complexcomprises double-stranded regions on either side of a single-strandedregion which includes a target site. In one embodiment, the target siteprobe includes three regions: a first region on the 5′ end of the targetsite probe is complementary to and hybridizes with the template sequenceupstream of a target site on the template sequence; a second region,which is 3′ of the first region, is non-complementary to the templatesequence and therefore does not hybridize with the template sequence;and a third region, which is on the 3′ end of the target site probe, iscomplementary to and hybridizes with the template sequence downstream ofthe target site. The target site probe can vary in nucleotide length,including but not limited to, about 5-19; about 20 to about 50nucleotides, about 51 to about 75 nucleotides, about 76 to about 100nucleotides and greater than 100 nucleotides.

[0157] Use of the target site probe directs the polymerase to aparticular enzyme binding site (i.e., the double-stranded segment andbubble formed upstream of the target site by the template sequence andthe primer) on the template sequence to facilitate the initiation oftranscription at a particular target site. That is, rather thanfacilitating the random initiation of synthesis reactions by thepolymerase along the length of a single-stranded template sequence, asdescribed above, this embodiment provides targeted binding of thepolymerase for the detection of a particular target site encompassed bythe bubble complex formed by the target site probe.

[0158] The target site probes used in the practice of the invention maybe made enzymatically or synthetically, using conventional nucleic acidsynthesis technology, such as phosphoramidite, H-phosphonate, orphosphotriester chemistry, for example. Alternative chemistries, such asthose which result in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, may also be employed.The target site probes may be ordered commercially from a variety ofcompanies which specialize in custom polynucleotides and/oroligonucleotides, such Operon, Inc. (Alameda, Calif.).

[0159] The sequence of the target site probe will vary depending uponthe target sequence. The overall length of the target site probe isselected to provide for hybridization of the first and third regionswith the target sequence and optimization of the length of the second,non-hybridized region. The first and third regions of the target siteprobe are designed to hybridize to known internal sites on the targetnucleic acid template. Depending upon the application, the sequence ofthe second region on the target site probe can be designed such that thesecond region may or may not be self-complementary. The overall lengthof the target site probe ranges from about 20 to about 50 nucleotides,preferably from about 25 to about 35 nucleotides. The first and thirdregions of the target site probe each range from about 5 to about 20nucleotides in length, preferably from about 8 to about 10 nucleotidesin length. In one embodiment, the first and third regions of the targetsite probe are each about 10 nucleotides in length. The internal, secondregion on the target site probe ranges in length from about 8 to about14 nucleotides, preferably from about 12 to about 14 nucleotides.

[0160] In one embodiment, at least one target site probe is used tospecifically initiate abortive oligonucleotide synthesis at one or moretarget sites on the nucleic acid template to produce multipleoligonucleotide products. In another embodiment, the target site probedirects the initiation of abortive transcription on a single-strandedtarget site in the absence of a template promoter sequence, as is knownin the art. (See, U.S. Pat. No. 5,571,669; Daube and von Hippel,Science, 258: 1320-1324 (1992)).

[0161] Abortive Promoter Cassette

[0162] In accordance with the invention, an abortive promoter cassette(APC) may be used to link a target to a defined sequence to generatemultiple detectable oligonucleotide products that indicate the presenceof the target in a test sample. The APC is a self-complementary sequenceof DNA that may consist of: (1) one contiguous oligonucleotide to whichRNA polymerase can bind to form a transcription bubble; (2) twopartially complementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; or (3) two complementaryoligonucleotides that form a transcription bubble region in the presenceof an RNA polymerase, which allows for the synthesis of an abortiveoligonucleotide product. The APC may contain an artificial promoter, orit may contain the promoter for a specific RNA polymerase. For example,trinucleotide or tetranucleotide products that could be generated fromwith a common phage RNA polymerase can be made with a labeled GpA orGpApA initiator and a labeled pppG or pppA terminator.

[0163] In an exemplary embodiment, as illustrated in FIG. 1, the APCcomprises eight regions, including an APC linker sequence whichcomprises either a 3′ or a 5′ single-stranded overhang region (i.e., a“sticky end”). A first region (A) on the 5′ end of the APC iscomplementary to a second region (A′) near the 3′ end of the APC. Athird region (B) and a fourth region (E) are separated from each otherby regions C, D, and C′ and are non-complementary to each other, suchthat the regions B and E form a single-stranded bubble region on the APCwhen the self-complementary regions of the APC interact with oneanother. Regions C and C′ are substantially self-complementary, suchthat the 5′ end of region C is complementary to the 3′ end of the regionC′. Region D may be a short sequence joining C and C′ for a contiguousAPC or may be a region comprising the free 3′ or 5′ ends of two separateupper and lower oligonucleotides for a two-part APC. Finally, the APCalso includes an APC linker, a single-stranded region on either the 5′end or the 3′ end of the APC oligonucleotide, which is formed throughthe complementary interaction of regions A and A′. The APC linkerfacilitates attachment of the APC with other target molecules, such ascaptured target DNA, RNA, or protein, for example.

[0164] The APC used in the practice of the invention may be madeenzymatically or synthetically, using conventional nucleic acidsynthesis technology, such as phosphoramidite, H-phosphonate, orphosphotriester chemistry, for example. Alternative chemistries, such asthose that result in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, may also be employed.The APC may be ordered commercially from a variety of companies thatspecialize in custom polynucleotides and/or oligonucleotides, such asOperon, Inc. (Alameda, Calif.).

[0165] The length of the APC is selected to optimize the stability ofthe bubble region and provide for the hybridization of the APC linkersequence with the target sequence. The overall length of the APC mayrange from about 50 to about 150 nucleotides, preferably from about 55to about 125 nucleotides. Regions A and A′ may each comprise from about5 to about 25 nucleotides and preferably comprise from about 7 to about15 nucleotides. Regions B and E may comprise from about 8 to about 16nucleotides and preferably comprise from about 10 to about 14nucleotides. Regions C and C′ may each comprise from about 5 to about 25nucleotides and preferably comprise from about 10 to about 20nucleotides. The single-stranded overhang region may comprise from about5 to about 40 nucleotides and preferably comprises from about 10 toabout 25 nucleotides.

[0166] Polymerase

[0167] Template-dependent polymerases for use in the methods andcompositions of the present invention are known in the art. Eithereukaryotic or prokaryotic polymerases may be used. In one embodiment,the template-dependent polymerase is a thermostable polymerase. Inanother embodiment, the polymerase is able to tolerate label moieties onthe phosphate group, the nuclease, and/or on the pentose ring ofunincorporated nucleotides. In one embodiment, the polymerase is aDNA-dependent RNA polymerase which is capable of transcribing asingle-stranded DNA template without a promoter sequence. In anotherembodiment, the polymerase is a DNA-dependent RNA polymerase which iscapable of transcribing a single-stranded DNA template having a promotersequence that is capable of binding the particular RNA polymerase beingused. In another embodiment, the polymerase is a DNA-dependent DNApolymerase that is capable of replicating a DNA target site to form aDNA oligonucleotide product. In a further embodiment, the polymerase isan RNA-dependent DNA polymerase that is capable of synthesizing asingle-stranded complementary DNA transcript from an RNA template.Examples of suitable polymerases include the RNA polymerases encoded byEscherichia coli, Escherichia coli bacteriophage T7, Escherichia colibacteriophage T3, and Salmonella typhimurium bacteriophage SP6;RNA-dependent RNA polymerases, such as poliovirus RNA polymerase;reverse transcriptases, such as HIV reverse transcriptase; and DNApolymerases such as Escherichia coli, T7, T4 DNA polymerase, Taqthermostable DNA polymerase, terminal transferase, primase, andtelomerase.

[0168] In general, the enzymes included in the methods of the presentinvention preferably do not produce substantial degradation of thenucleic acid components produced by the methods.

[0169] Nucleotides

[0170] In accordance with the invention, the polymerase catalyzes areaction in the usual 5′→3′ direction on the oligonucleotide product andeither transcribes or replicates the target nucleic acid by extendingthe 3′ end of the initiator or primer through the sequential addition ofnucleotides (NTPs), which may include nucleotide analogs (NTP analogs)and which may be labeled or unlabeled. To facilitate reiterative,abortive synthesis initiation events, the NTPs and/or NTP analogs thatare added to the reaction mixture before and/or during the synthesisreaction include a chain terminator, which is capable of terminating thesynthesis event initiated by the polymerase. Use of the chain terminatorstalls the polymerase during the synthesis reaction, inhibits formationof a processive elongation complex, and thereby promotes the reiterativesynthesis of short abortive oligonucleotides from the target site.(Daube and von Hippel, Science, 258: 1320-1324 (1992)).

[0171] In accordance with the invention, a chain terminator may compriseany compound, composition, complex, reactant, reaction condition, orprocess step (including withholding a compound, reactant, or reactioncondition) which is capable of inhibiting the continuation oftranscription or replication by the polymerase during the primerextension reaction. In one embodiment, a suitable chain terminator isNTP deprivation, that is, depriving the polymerase of the particular NTPthat corresponds to the subsequent complementary nucleotide of thetemplate sequence. In other words, since NTP requirements for chainelongation are governed by the complementary strand sequence, given adefined template sequence and a defined primer length, a selected NTPmay be withheld from the reaction mixture such that termination of chainelongation by the polymerase results when the reaction mixture fails toprovide the polymerase with the NTP that is required to continuetranscription or replication of the template sequence.

[0172] Alternatively, in another embodiment, the chain terminator mayinclude nucleotide analogs, which may be labeled or unlabeled and which,upon incorporation into an oligonucleotide product by the polymerase,effect the termination of nucleotide polymerization. Specifically, sincechain elongation by a polymerase requires a 3′ OH for the addition of asubsequent nucleotide, nucleotide analogs having a suitably modified 3′end will terminate chain elongation upon incorporation into theoligonucleotide product. Nucleotide analogs having chain terminatingmodifications to the 3′ carbon of the pentose sugar are known in the artand include nucleotide analogs such as 3′ dideoxyribonucleosidetriphosphates (ddNTPs) and 3′ O-methylribonucleoside 5′ triphosphates,as well as nucleotide analogs having either a —H or a —OCH₂ moiety onthe 3′ carbon of the pentose ring. Alternatively, in a furtherembodiment, the chain terminator may include nucleotide analogs, eitherlabeled or unlabeled, which have a 3′ OH group, but which, uponincorporation into the oligonucleotide product, still effect chaintermination at some positions, as described herein (Costas, Hanna, etal., Nucleic Acids Research 28: 1849-58 (2000); Hanna, M., MethEnzymology 180: 383-409 (1989); Hanna, M., Nucleic Acids Research 21:2073-79 (1993); Hanna, M. et al., Nucleic Acid Research 27: 1369-76(1999)).

[0173] NTPs and/or NTP analogs that can be employed to synthesizeabortive oligonucleotide products in accordance with the methods of theinvention may be provided in amounts ranging from about 1 to about 5000μM, preferably from about 10 to about 2000 μM. In a preferred aspect,nucleotides and/or nucleotide analogs, such as ribonucleosidetriphosphates or analogs thereof, that can be employed to synthesizeoligonucleotide RNA transcripts by the methods of the invention may beprovided in amounts ranging from about 1 to about 6000 μM, preferablyfrom about 10 to about 5000 μM.

[0174] Labeling and Detection

[0175] In accordance with an aspect of the invention, detectableoligonucleotide products are synthesized from a target nucleic acidtemplate. The detection and identification of the oligonucleotideproducts are facilitated by label moieties on the initiator and/or onthe NTPs or NTP analogs that are incorporated by the polymerase intoeach oligonucleotide product that is synthesized on the target nucleicacid and/or on other molecules which are part of the synthetic complexor which interact with one or more components of the synthetic complex.The label or reporter moieties may be chemically or enzymaticallyincorporated into the nucleotides forming the primer and/or into thereactant NTPs or NTP analogs that are utilized by the polymerase duringthe extension reaction, or other molecules, and may include, forexample, fluorescent tags; paramagnetic groups; chemiluminescent groups;metal binding sites; intercalators; photochemical crosslinkers;antibody-specific haptens; metals; small molecules which are members ofa specific binding pair (such as biotin and streptavidin for example);and any other reporter moiety or moieties which can produce a detectableand/or quantifiable signal either directly or indirectly. Exemplarynucleotide analogs may include, for example, 8-modified purines(8-APAS-ATP) (Costas, Hanna, et al., Nucleic Acids Research 28: 1849-58(2000)); 5-modified pyrimidines (5-APAS-UTP; 5-APAS-CTP) (Hanna, M.,Meth Enzymology 180: 383-409 (1989); Hanna, M., Nucleic Acids Research21: 2073-79 (1993)); fluorescent ribonucleotides (5-SF-UTP) (Hanna, M.et al., Nucleic Acid Research 27: 1369-76 (1999)); and hapten-taggeddeoxynucleotide precursors (5-DNP-SdU) (Meyer and Hanna, BioconjugateChem 7: 401-412 (1996); U.S. Pat. Nos. 6,008,334 and 6,107,039).

[0176] In one embodiment, a fluorophore moiety is attached to the 5′ endof the initiator that is used to initiate transcription of the targetnucleic acid. In another embodiment, a fluorophore moiety is attached tothe 5 or 8 position of the base of an NTP or NTP analog that is used bythe polymerase to extend the initiator primer. In a further embodiment,a first fluorophore moiety is attached to the initiator and a secondfluorophore is attached to an NTP or NTP analog that is used to extendthe initiator. In this latter embodiment, a fluorescent energy transfermechanism can be used, wherein the first fluorophore (e.g. fluorescein,aedans, coumarin, etc.) is excited and the emission is read from thesecond fluorophore (e.g. fluorescein, aedans, coumarin, etc.) when thesecond fluorophore is brought into proximity with the first fluorophoreby the polymerase during synthesis of the oligonucleotide product.Alternatively, the first and second fluorophores may function by anelectron transfer mechanism, wherein the first fluorophore absorbsenergy from the second fluorophore when the polymerase brings the firstand second fluorophores into proximity with each other, and the firstfluorophore releases the energy in a radiative manner, thereby enablingdetection.

[0177] In one aspect, a first fluorophore is a fluorescent energy donor,which is attached to a first reactant (i.e., either a nucleotide that isincorporated into the initiator or a nucleotide that is to beincorporated by the polymerase into the oligonucleotide product), and asecond fluorophore is a fluorescent energy acceptor, which is attachedto a second reactant (either a nucleotide that is incorporated into theinitiator nucleotide or a nucleotide that is to be incorporated by thepolymerase into the oligonucleotide product) that is different from thefirst reactant. In one embodiment, each of the four NTPs or NTP analogsthat may be used to extend the primer is tagged with a uniquefluorescent energy acceptor which is capable of a distinct emissionwavelength when brought into proximity with the fluorescent energy donoron the primer. Preferably, the fluorescent energy transfer can bemeasured in real time, without isolation of the oligonucleotideproducts, since neither the initiator nor unincorporated NTPs or NTPanalogs alone will produce a signal at the wavelength used fordetection.

[0178] Fluorescent and chromogenic molecules and their relevant opticalproperties are amply described in the literature. See, for example,Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2ndEdition (Academic Press, New York, 1971); Griffiths, Colour andConstitution of Organic Molecules (Academic Press, New York, 1976);Bishop, ed., Indicators (Pergamon Press, Oxford, 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence(Interscience Publishers, New York, 1949); and the like. Further, thereis extensive guidance in the literature for derivatizing fluorophore andquencher molecules for covalent attachment via common reactive groupsthat can be added to a nucleotide, as exemplified by the followingreferences: Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345;Khanna et al., U.S. Pat. No. 4,351,760; Costas, Hanna, et al., NucleicAcids Research 28: 1849-58 (2000); Hanna, M. et al., Nucleic AcidResearch 27: 1369-76 (1999); and Meyer and Hanna, Bioconjugate Chem 7:401-412 (1996).

[0179] In general, nucleotide labeling can be accomplished through anyof a large number of known nucleotide labeling techniques using knownlinkages, linking groups, and associated complementary functionalities.Suitable donor and acceptor moieties that can effect fluorescenceresonance energy transfer (FRET) include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)amninonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate; erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B, sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.

[0180] There are many linking moieties and methodologies for attachingfluorophores to nucleotides, as exemplified by the following references:Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRLPress, Oxford, 1991); Zuckerman et al., Nucleic Acids Research 15:5305-5321(1987) (3′ thiol group on oligonucleotide); Sharma et al.,Nucleic Acids Research 19: 3019 (1991) (3′ sulfhydryl); Giusti et al.,PCR Methods and Applications 2: 223-227 (1993); Fung et al., U.S. Pat.No. 4,757,141 (5′ phosphoamino group via Aminolink™ II, available fromApplied Biosystems, Foster City, Calif.); Stabinsky, U.S. Pat. No.4,739,044 (3′ aminoalkylphosphoryl group); Agrawal et al., TetrahedronLetters 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research 15: 4837 (1987) (5-mercaptogroup); Nelson et al., Nucleic Acids Research 17: 7187-7194 (1989) (3′amino group); Hanna, M., Meth Enzymology 180: 383-409 (1989); Hanna, M.,Nucleic Acids Research 21: 2073-79 (1993); Hanna, M. et al., NucleicAcid Research 27: 1369-76 (1999) (5-mercapto group); Costas, Hanna, etal., Nucleic Acids Research 28: 1849-58 (2000) (8-mercapto group); andthe like.

[0181] In accordance with the invention, detection of theoligonucleotide products is indicative of the presence of the targetsequence. Quantitative analysis is also feasible. Direct and indirectdetection methods (including quantitation) are well known in the art.For example, by comparing the amount of oligonucleotide products thatare generated from a test sample containing an unknown amount of atarget nucleic acid to an amount of oligonucleotide products that weregenerated from a reference sample that has a known quantity of a targetnucleic acid, the amount of a target nucleic acid in the test sample canbe determined. The reiterative abortive synthesis initiation anddetection methods of the present invention can also be extended to theanalysis of genetic sequence alterations in the target nucleic acid, asfurther described below.

[0182] Reaction Conditions

[0183] Most transcription reaction conditions are designed for theproduction of full length transcripts, although no conditions have beenidentified that eliminate abortive transcription. Appropriate reactionmedia and conditions for carrying out the methods of the presentinvention include an aqueous buffer medium that is optimized for theparticular polymerase. In general, the buffer includes a source ofmonovalent ions, a source of divalent cations, and a reducing agent,which is added to maintain sulfhydral groups in the polymerase in areduced form. Any convenient source of monovalent ions, such as KCl,K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, ammonium sulfate, and thelike, may be employed. The divalent cation may be magnesium, managanese,zinc, or the like, though, typically, the cation is magnesium (Mg). Anyconvenient source of magnesium cations may be employed, including MgCl₂,Mg-acetate, and the like. The amount of Mg²⁺ present in the buffer mayrange from about 0.5 to 20 mM, preferably from about 1 to 12 mM.

[0184] Representative buffering agents or salts that may be present inthe buffer include Tris Phosphate, Tricine, HEPES, MOPS, and the like,where the amount of buffering agent typically ranges from about 5 to 150mM, usually from about 10 to 100 mM, and preferably from about 20 to 50mM. In certain embodiments, the buffering agent is present in an amountsufficient to provide a pH ranging from about 6.0 to 9.5, preferablyranging from about 7.0 to 8.0. Other agents which may be present in thebuffer medium include chelating agents, such as EDTA, EGTA, and thelike, or other polyanionic or cationic molecules (heparins, spermidine),protein carriers (BSA) or other proteins, including transcriptionfactors (sigma, NusA, Rho, lysozyme, GreA, GreB, NusG, etc.).

[0185] Variations in all of the reaction components potentially canalter the ratio of abortive transcripts to full-length transcripts.Alterations in the concentration of salts (from 10 mM to 100 mM) or theuse of alternative monovalent cations (K⁺ versus Na⁺ versus Rb⁺) havebeen shown to affect the level of transcription (measured as abortivetranscription) on linear DNA templates (Wang, J-Y, et al., Gene196:95-98 (1997)). Alternative sulfhydral reducing reagents are reportedto have differential effects on abovtive transcription.2-mercaptoethanol at 1-2 mM is reported to enhance abortivetranscription on a poly[dA-dT] template compared to the alternativereducing agent 5,5′-dithio-bis-(2-nitrobenzoic) acid (Job, D., ActaBiochem. Pol. 41:415-419 ((1994)).

[0186] A high molar ratio of RNA polymerase to template enhances thefrequency of abortive transcription over full length transcription onthe lambda P_(R) promoter. This effect apparently arises from collisionsbetween tandem polymerases at the promoter.

[0187] Certain RNA polymerase mutants have elevated rates of abortivetranscription compared to the wild-type polymerase. For example, amutation changing an arginine to a cysteine at codon 529 in the RNApolymerase beta subunit gene causes elevated abortive transcriptioin atthe E. coli pyrB1 promoter (Jin, D. J. and Turnbough, Jr., C. L., J.Mol. Biol. 236:72-80 (1994)).

[0188] The relative level of abortive transcription is sensitive to thenucleotide sequence of the promoter. A number of promoters have beenidentified that are unusually susceptible to abortive transcription(e.g., the galP2 promoter). The assay system that relies on recruitmentof a defined promoter can be optimized by screening candidate promotersfor maximal initiation frequency and maximal proportion of abortivetranscripts.

[0189] Any aspect of the methods of the present invention can occur atthe same or varying temperatures. In one embodiment, the reactions areperformed isothermally, which avoids the cumbersome thermocyclingprocess. The synthesis reaction is carried out at a temperature thatpermits hybridization of the various oligonucleotides, including targetsite probes, capture probes, and APCs, as well as the primers to thetarget nucleic acid template and that does not substantially inhibit theactivity of the enzymes employed. The temperature can be in the range ofabout 25° C. to about 85° C., more preferably about 30° C. to about 75°C., and most preferably about 25° C. to about 55° C. In someembodiments, the temperature for the transcription or replication stepsmay be different than the temperature(s) for the preceding steps. Thetemperature of the transcription or replication steps can be in therange of about 25° C. to about 85° C., more preferably about 30° C. toabout 75° C., and most preferably about 25° C. to about 55° C.

[0190] Denaturation of the target nucleic acid in a test sample may benecessary to carry out the assays of the present invention in caseswhere the target nucleic acid is found in a double-stranded form or hasa propensity to maintain a rigid structure. Denaturation is a processthat produces a single-stranded nucleic acid and can be accomplished byseveral methods that are well-known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (New York, Cold SpringHarbor Laboratory Press, third edition, 2000). One method for achievingdenaturation includes the use of heat, such as exposing the nucleic acidin a test sample to temperatures of about 90-100° C. for about 2-20minutes. Alternatively, a base may be used as a denaturant when thenucleic acid comprises DNA. Many basic solutions, which are well-knownin the art, may be used to denature a DNA sample. An exemplary methodincubates the DNA sample with a base, such as NaOH for example, at aconcentration of about 0.1 to 2.0 N NaOH at a temperature ranging fromabout 20° C. to about 100° C. for about 5-120 minutes. Treatment with abase, such as sodium hydroxide, not only reduces the viscosity of thesample, which increases the kinetics of subsequent enzymatic reactions,but also aids in homogenizing the sample and reducing the possibility ofbackground by destroying any existing DNA-RNA or RNA-RNA hybrids thatmay exist in the sample.

[0191] In accordance with various aspects and embodiments of theinvention, the target nucleic acid molecules may be hybridized to anoligonucleotide capture probe, a mononucleotide or oligonucleotideinitiator which is complementary to a portion of the target nucleicacid, an APC linker sequence that is complementary to a portion of atarget nucleic acid, and/or a target site probe that is complementary toregions on either side of the target site. Hybridization is conductedunder standard hybridization conditions that are well-known to thoseskilled in the art. Reaction conditions for hybridization of anoligonucleotide (or polynucleotide) to a target sequence vary fromoligonucleotide to oligonucleotide, depending upon factors such asoligonucleotide length, the number of G:C base pairs present in theoligonucleotide, and the composition of the buffer utilized in thehybridization reaction. Moderately stringent hybridization conditionsare generally understood by those skilled in the art to be conditionsthat are approximately 25° C. below the melting temperature of aperfectly base-paired double-stranded DNA. Higher specificity isgenerally achieved by employing more stringent conditions, such asincubation conditions having higher temperatures. Chapter 11 of thewell-known laboratory manual of Sambrook et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory Press, 1989)describes hybridization conditions for oligonucleotide probes andprimers in great detail, including a description of the factors involvedand the level of stringency necessary to achieve hybridization with thedesired degree of specificity.

[0192] The oligonucleotide capture probe, the target site probe, theAPC, and/or the initiator may each be incubated with the target nucleicacid for about 5 to 120 minutes at about 20 to 80° C. to permithybridization. Preferably, the target nucleic acid and theoligonucleotide probes, the APC, and/or the initiator are incubated forabout 5 to 60 minutes at about 25 to 70° C. More preferably, the targetnucleic acid and the oligonucleotide probes, the APC, and/or primer areincubated for about 5-30 minutes at about 35-50° C.

[0193] Hybridization is typically performed in a buffered aqueoussolution and temperature conditions, salt concentration, and pH areselected to provide sufficient stringency to enable the oligonucleotideprobes, the APC, or the primer to hybridize specifically to the targetsequence but not to any other sequence. Generally, the efficiency ofhybridization between an oligonucleotide or polynucleotide and a targetnucleic acid template will be improved under conditions where the amountof oligonucleotide or polynucleotide added to the reaction mixture is inmolar excess to the template, preferably a molar excess that ranges fromabout 10³ to 10⁶. It will be appreciated, however, that the amount oftarget nucleic acid in the test sample may not be known, so that theamount of an oligonucleotide, such as the amount of an oligonucleotidecapture probe, a target site probe, or an APC for example, relative toan amount of a target nucleic acid template cannot be determined withcertainty.

[0194] Alternatively, if a target DNA sequence has been treated with abase to effect denaturation, the oligonucleotide or polynucleotide isdiluted in a probe diluent that also acts as a neutralizinghybridization buffer. In this manner, the pH of the test sample can bekept between about 6 and 9, which will favor the hybridization reactionand will not interfere with subsequent enzymatic reactions. Preferably,the neutralizing buffer is a 2-[bis(2-hydroxyethyl) amino]ethanesulfonic acid (“BES”) (Sigma, St. Louis, Mo.) and sodium acetate buffer.More preferably, the neutralizing hybridization buffer is a mixture of 2M BES, 1 M sodium acetate, 0.05% of an antimicrobial agent, such asNaN₃, 5 mM of a chelating agent, such as EDTA, 0.4% of a detergent, suchas Tween-20™, and 20% of a hybridization accelerator, such as dextransulfate. The pH of the neutralizing hybridization buffer is betweenabout 5 to 5.5.

[0195] Transcription conditions and reagents are well-known in the art.Examples of typical conditions and reagents for RNA polymerasetranscription and DNA polymerase replication are readily found in theliterature. See, e.g., Chamberlain et al., The Enzymes, Boyer, ed., NewYork Acad. Press, 3rd ed., p. 85 (1982); Dunn et al., M. Mol. Biol. 166:477-535 (1983)); Geider, Proc. Natl. Acad. Sci. USA 75: 645-649 (1978));Guruvich et al., Analytical Biochem 195: 207-213 (1991); Lewis et al.,J. Biol. Chem. 255: 4928-4936 (1980); Martin et al., Biochem. 27:3966-3974 (1988); and Milligan et al., Methods Enzymol. Vol. 180a, ed.,50-52 (1989)). As described in Lu et al., U.S. Pat. No. 5,571,669,polymerase concentrations for transcription initiated from artificialtranscription bubble complexes are generally about one order ofmagnitude higher than the ideal polymerase concentrations forpromoter-initiated, or palindromic sequence-initiated, transcription.

[0196] In one embodiment, the foregoing components are addedsimultaneously at the initiation of the abortive synthesis and detectionmethods. In another embodiment, components are added in any order priorto or after appropriate timepoints during the method, as required and/orpermitted by the various reaction steps. Such timepoints can be readilyidentified by a person of skill in the art. The enzymes used for nucleicacid detection according to the methods of the present invention can beadded to the reaction mixture prior to or following the nucleic aciddenaturation step, prior to or following hybridization of the primer tothe target nucleic acid, prior to or following the optionalhybridization of the target site probe to the target nucleic acid, orprior to or following the optional hybridization of the APC, asdetermined by the enzymes' thermal stability and/or other considerationsknown to those skilled in the art.

[0197] The various reaction steps in the methods of the invention can bestopped at various timepoints and then resumed at a later time. Thesetimepoints can be readily identified by a person of skill in the art.Methods for stopping the reactions are known in the art, including, forexample, cooling the reaction mixture to a temperature that inhibitsenzyme activity. Methods for resuming the reactions are also known inthe art, including, for example, raising the temperature of the reactionmixture to a temperature that permits enzyme activity. In someembodiments, one or more of the components of the various reactions maybe replenished prior to, at the time of, or following the resumption ofthe reactions. Alternatively, the reaction can be allowed to proceed(i.e., from start to finish) without interruption.

[0198] Abortive Synthesis and Detection Methods of the Invention

[0199] The following examples of the abortive synthesis and detectionmethods of the invention are provided to more specifically describe theinvention. These exemplary methods are intended to be merelyillustrative and are not intended to limit the description providedabove. It will be appreciated that various other embodiments may bepracticed, given the above general description. For example, referenceto the use of a primer means that any of the primers described hereinmay be used, including RNA initiators.

[0200] In accordance with an aspect of the invention, a method fordetecting the presence of a target polynucleotide by generating multipledetectable oligonucleotide products through reiterative synthesisinitiation events on the target polynucleotide is provided. FIG. 2diagrammatically illustrates the various reactants that may be combinedand reacted in the presence of RNA polymerase to synthesize multipledetectable oligonucleotide products. The methods of the invention may beperformed using a test sample that potentially contains a targetsequence. The test sequence may be detected directly or the product ofprimer-extension or reverse transcription of the target may be detected.Sequences or tags may be added to the copy of the target (e.g., biotin,ssDNA regions). The test sample may include double-stranded DNA,single-stranded DNA, or RNA. The DNA or RNA may be isolated and purifiedby standard techniques for isolating DNA or RNA from cellular, tissue,or other samples. Such standard methods may be found in references suchas Sambrook et al., Molecular Cloning: A Laboratory Manual (New York,Cold Spring Harbor Laboratory Press, third edition, 2000). In oneembodiment, the target nucleic acid is DNA or RNA that is in a suitablemedium, although the target nucleic acid can be in lyophilized form.Suitable media include, but are not limited to, aqueous media (such aspure water or buffers). In another embodiment, the target nucleic acidis immobilized prior to being utilized as a substrate for a synthesisreaction.

[0201] In an exemplary embodiment, the target sequence is immobilized bya sequence-specific (e.g., gene-specific) oligonucleotide capture probethat is attached to a solid matrix, such as a microtiter plate. Theimmobilized capture probe is treated under hybridizing conditions with atest sample that includes single-stranded DNA (i.e., denatured DNA) orRNA. Any target sequence that is present in the test sample hybridizesto the capture probe and is then exposed to additional reagents inaccordance with the invention.

[0202] In an exemplary embodiment, an initiator (n 5′-R₁—(N_(I))_(x)—OH3′) hybridizes with the target sequence upstream of a target site in thepresence of the target site probe (FIG. 11) and facilitates catalysis ofa polymerization reaction at the target site by the polymerase. Theinitiation primer may be comprised of nucleosides, nucleoside analogs,nucleotides, and nucleotide analogs. The initiaor primer may vary in thenumber of nucleotides, such as nucleotides from 1-25 nucleotides, 26-50nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides,126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225nucleotides, 226-250 nucleotides, and greater than 250 nucleotides, andmay include one or more nucleotide analogs. A suitable RNA polymerase isemployed to synthesize an oligoribonucleotide product from the targetsequence or any portion thereof. The polymerase may be an RNA-dependentor DNA-dependent RNA polymerase. The DNA or RNA target sequence may ormay not be attached to other molecules, such as proteins, for example.

[0203] During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotideswhich have been added to the reaction mixture. As the polymerasereaction proceeds, the polymerase extends the initiator, as directed bythe template sequence, by incorporating corresponding nucleotides thatare present in the reaction mixture. In one embodiment, these reactantnucleotides comprise a chain terminator (e.g., n 5′ pppN_(T)—R₂, a chainterminating nucleotide analog, as described above). When the polymeraseincorporates a chain terminator into the nascent oligonucleotideproduct, chain elongation terminates due to the polymerase's inabilityto catalyze the addition of a nucleotide at the 3′ position on thepentose ring of the chain terminator. Consequently, the polymeraseaborts the initiated synthesis event by releasing the oligonucleotideproduct (i.e., 5′ R₁—(N_(I))_(z)pN_(T)—R₂, where z=x+y) and reinitiatingthe abortive initiation synthesis reaction at the target site.

[0204] The abortive initiation reaction may be controlled such that thepolymerase aborts synthesis after extending the initiator by apredetermined number of nucleotides. For example, if it is desirable toterminate the synthesis reaction after the initiator has been extendedby a single nucleotide, this may be accomplished by, for example,either: (1) adding to the reaction mixture only nucleotides that arechain terminators, thereby inhibiting polymerization after the firstnucleotide is incorporated by the polymerase; or (2) if the geneticsequence of the target site is known, adding to the reaction mixtureonly a preselected chain terminating nucleotide analog (i.e., nucleotideanalogs which comprise one of A, G, T, C, or U) that is complementary tothe nucleotide at the target site. Alternatively, if it is desirable toterminate the synthesis reaction after the initiator has been elongatedby a predetermined number of nucleotides, and if the genetic sequence ofthe target site is known, this may be accomplished by, for example,adding to the reaction mixture a preselected chain terminatingnucleotide analog (i.e., nucleotide analogs which comprise one of A, G,T, C, or U) that is complementary to an Nth nucleotide from the targetsite, where N is the predetermined number of nucleotides comprised bythe oligonucleotide product, exclusive of the initiator. In this manner,multiple abortive oligonucleotide products that comprise the initiatorand a chain terminating nucleotide analog are synthesized by thepolymerase.

[0205] The polymerase releases the oligonucleotide product withouttranslocating from the enzyme binding site or dissociating from thetarget polynucleotide sequence. Nucleotide deprivation can be used tosequester the polymerase at the polymerase binding site. For example, ifonly an initiator and a terminator are supplied, elongation by thepolymerase will not be possible.

[0206] Furthermore, reaction conditions may be optimized for abortivetranscription initiation, whereby it is favorable for the polymerase toremain bound to the polymerase binding site even in the presence ofelongating nucleotides. The abortive initiation reaction buffer will beoptimized to increase the abortive events by adjusting theconcentrations of the salts, the divalent cations, the glycerol content,and the amount and type of reducing agent to be used. In addition,“roadblock” proteins may be used to prevent the polymerase fromtranslocating.

[0207] In another aspect of the invention, the initiator includes amoiety (e.g., R₁, as depicted in FIG. 2) which may be covalently bondedto the 5′ phosphate group (as in FIG. 3), the 2′ position of the pentosering, or the purine or pyrimidine base of one of the nucleotides ornucleotide analogs that are incorporated into the initiator.Additionally, the reactant nucleotides and/or nucleotide analogs thatare included in the reaction mixture for incorporation into theoligonucleotide product by the polymerase may each also include a moiety(e.g., R₂, as depicted in FIG. 2), which is covalently bonded to eitherthe nucleobase (as in FIG. 4) or the 2′ position or 3′ position of thepentose ring. In an exemplary embodiment, R₁ and R₂ are label moieties(as in FIG. 5) on the initiator and the chain terminator, respectively,that are incorporated into the oligonucleotide product by the polymerase(as in FIG. 6) and are adapted to interact in a manner that generates adetectable signal (e.g., fluorescence resonance energy transfer (FRET)(FIG. 7), fluorescence or colorimetry (FIG. 8)), thereby permitting thedetection and quantitation of the synthesized oligonucleotide products.In one embodiment, as illustrated in FIG. 9, an oligonucleotide product(5′ R₁—(N_(I))_(x)pN_(T)—R₂) incorporating an initiator (N_(I)) that hasan energy donor group (R₁) and a chain terminating nucleotide (N_(T))that has an energy acceptor group (R₂) generates a signal throughfluorescence resonance energy transfer from R₁ to R₂ when thesynthesized oligonucleotide products are irradiated with light of aparticular wavelength. As shown in FIG. 9, when the energy donor moietyR₁ on the initiator is excited by exposure to light of a specifiedwavelength (λ_(1A)) (e.g., the absorption maximum of R₁) the exciteddonor moiety R₁ emits light of a second wavelength (λ_(1E/2A)) (e.g.,the emission maximum for R₁) which is absorbable by R₂. If N_(T) hasbeen suitably incorporated into the oligonucleotide product by thepolymerase, the energy acceptor moiety R₂ on N_(T) is positionedsufficiently near R₁ on N_(I) (e.g., within about 80 Å) to facilitateefficient energy transfer between R₁ and R₂, such that R₂ absorbs thewavelength of light (λ_(1E/2A)) emitted by the excited donor moiety R₁.In response to the absorption of λ_(1E/2A), the excited R₂ acceptormoiety emits light of a third wavelength (λ_(2E)), which may then bedetected and quantified in accordance with methods that are well-knownin the art. Exemplary R₁ and/or R₂ FRET label moieties include aedansand fluorescein (as shown in FIG. 7), or pyrene, stilbene, coumarine,bimane, naphthalene, pyridyloxazole, naphthalimide, NBD, BODIPY™, aswell as any of those described in greater detail above.

[0208] In an alternate embodiment, as diagrammatically illustrated inFIG. 10, n copies of a dinucleotide initiator (5′R₁—N₁pN₂—R₂—OH 3′)comprising reporter moieties (R₁ and R₂) on either of the nucleotides(N₁ and N₂, respectively) may be extended by a polymerase to incorporaten copies of a chain terminator (5′ pppN₃R₃) which includes a thirdreporter moiety (R₃), yielding n copies of a detectable trinucleotidetranscript (5′ R₁—N₁pN₂R₂pN₃-R₃—OH 3′). In a manner similar to the onedescribed above with reference to FIG. 10, the trinucleotide transcriptmay be irradiated with a first wavelength of light (λ_(1A)) whichexcites the R₁ energy donor group on the first nucleotide (N₁) to emitλ_(1E)/λ_(3A). λ_(1E)/λ_(3A) is then absorbed by the R₃ energy acceptorgroup on the chain terminating nucleotide (N₃), and an excited R₃ thenemits λ_(3E), which can then be detected and quantified. Alternatively,the transcript may be irradiated with a second wavelength of light(λ_(2A)) which excites an R₂ energy donor group on a second nucleotide(N₂) to emit λ_(2E)/λ_(3A). λ_(2E)/λ_(3A) is then absorbed by the R₃energy acceptor group, and an excited R₃ then emits λ_(3E), which can bedetected and quantified. In either case, the detectable wavelength(λ_(3E)) is not obtained unless the polymerase brings an energy donorreporter moiety on the initiator (R₁ or R₂) into sufficient proximitywith a corresponding energy acceptor reporter moiety (R₃) on theincorporated nucleotide to result in the emission of the detectablewavelength of light.

[0209] In another aspect of the invention, as diagrammaticallyillustrated in FIG. 11, a target site probe may be used to form a bubblecomplex in a target region of the target sequence. As described above,the bubble complex comprises double-stranded regions that flank asingle-stranded region that includes a target site. In this embodiment,the target site probe is used to direct the polymerase to the targetsite by positioning the target site at the junction of thesingle-stranded bubble region and a downstream duplex region on thetarget sequence. In an exemplary embodiment, the target site probecomprises from about 18-54 nucleotides: a first region (A) whichhybridizes to the target sequence (A′) upstream of the target sitecomprises about 5-20 nucleotides; an internal, second region ofnon-base-paired nucleotides (B) comprises about 8-14 nucleotides; and athird region (C) which hybridizes to the target sequence downstream ofthe target site (C′) comprises about 5-20 nucleotides. The polymeraseassociates with an initiator and initiates a synthesis reaction at thetarget site on the template sequence. The polymerase elongates theinitiator to synthesize an abortive oligonucleotide product through theincorporation of nucleotides, which comprise a suitable chainterminator. Both the initiator and the nucleotides, including the chainterminating nucleotide, may be modified with a label moiety to allowsignal detection, such as by fluorescence resonance energy transfer forexample, as described above.

[0210] An illustrative procedure for detecting multiple oligonucleotideproducts through reiterative synthesis initiation events on a targetsequence, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isdesigned to hybridize with a specific or general target sequence; (b)optionally hybridizing the oligonucleotide capture probe with a testsample which potentially contains a target sequence; (c) optionallyhybridizing the target sequence with a target site probe; (d) modifyingat least one of an initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide productsynthesized by the polymerase; (e) hybridizing the target sequence withthe primer; and (f) extending the initiator with a polymerase such thatthe polymerase reiteratively synthesizes an oligonucleotide product thatis complementary to a target site by incorporating complementarynucleotides comprising a chain terminator and releasing an abortiveoligonucleotide product without either translocating from an enzymebinding site or dissociating from the target sequence.

[0211] During transcription of the template by the RNA polymerase, theRNA initiator is extended by the RNA polymerase through theincorporation of nucleotides that have been added to the reactionmixture. As the polymerase reaction proceeds, the RNA polymerase extendsthe RNA initiator, as directed by the template sequence, byincorporating corresponding nucleotides that are present in the reactionmixture. In one embodiment, these reactant nucleotides comprise a chainterminator (e.g., n 5′ pppN_(T)-R₂, a chain terminating nucleotideanalog, as described above). When the RNA polymerase incorporates achain terminator into the nascent transcript, chain elongationterminates due to the polymerase's inability to catalyze the addition ofa nucleotide at the 3′ position on the ribose ring of the chainterminator, and the RNA polymerase aborts the initiated transcriptionevent by releasing the transcript and reinitiating transcription at thetarget site. The abortive transcription initiation reaction may becontrolled such that multiple abortive oligonucleotide transcripts of apredetermined length and comprising the RNA primer and a chainterminating nucleotide analog are generated.

[0212] In an exemplary embodiment, the RNA initiator may be amononucleotide and the nucleotides provided in the reaction mixture maycomprise solely chain terminators. In this embodiment, transcription isaborted by the RNA polymerase after the RNA initiator has been extendedby a single nucleotide and an abortive dinucleotide transcript isgenerated. In another embodiment, the RNA initiator may comprise adinucleotide or a trinucleotide, for example, and an abortivetranscription initiation event may generate an abortive transcriptcomprising a trinucleotide or a tetranucleotide, respectively. It willbe appreciated that abortive transcripts of any desired length may beobtained, depending upon the length of the RNA initiator and the natureand composition of the reactant nucleotides that are selected forinclusion in the reaction mixture. For example, if the nucleotidesequence of the template is known, the components (e.g., target site,initiator, and reactant nucleotides) of the transcription reaction maybe selected such that abortive transcripts of any desired length aregenerated by the method of the invention.

[0213] In another aspect of the invention, the RNA initiator includes amoiety (e.g., R₁, as depicted in FIG. 6) which may be covalently bondedto the 5′ phosphate group, the 2′ position of the ribose ring, or thepurine or pyrimidine base of one of the nucleotides or nucleotideanalogs that are incorporated into the RNA initiator. Additionally, thereactant nucleotides and/or nucleotide analogs that are included in thereaction mixture for incorporation into the oligonucleotide transcriptby the RNA polymerase may each also include a moiety (e.g., R₂, asdepicted in FIG. 6), which is covalently bonded to either the nucleobaseor the 2′ position or 3′ position of the ribose ring. The moieties R₁and R₂ may each comprise H, OH, or any suitable label moiety, reportergroup, or reporter group precursor, as described in greater detailabove.

[0214] An illustrative procedure for detecting multiple oligonucleotidetranscripts through reiterative transcription initiation events on atarget sequence, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isdesigned to hybridize with a specific or general target sequence; (b)optionally hybridizing the oligonucleotide capture probe with a testsample which potentially contains a target sequence; (c) optionallyhybridizing the target sequence with a target site probe; (d) modifyingat least one of an RNA initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide transcriptsynthesized by the RNA polymerase; (e) hybridizing the target sequencewith the RNA initiator; and (f) extending the RNA initiator with an RNApolymerase such that the RNA polymerase reiteratively synthesizes anoligonucleotide transcript that is complementary to a target site byincorporating complementary nucleotides comprising a chain terminatorand releasing an abortive oligonucleotide transcript withoutsubstantially translocating from the polymerase binding site ordissociating from the target sequence.

[0215] In accordance with another aspect of the invention, asdiagrammatically illustrated in FIG. 8, the methods of the invention maybe utilized to generate an oligonucleotide product (5′R₁—(N_(I))_(x)pN_(T)—R₂) which comprises an initiator (N_(I)) with amoiety (R₁), such as an immobilization tag for example; and a chainterminating nucleotide (N_(T)) that includes a label moiety (R₂), suchas a signal generator or signal generator precursor for example. In thisembodiment, the oligonucleotide product(s) may be captured orimmobilized, such as on a membrane for example, to facilitate detectionof the oligonucleotide products of the abortive synthesis reaction. Inan exemplary embodiment, R₁ is a bioadhesive tag, such as biotin forexample; R₂ is a label moiety, such as fluorescein for example; andoligonucleotide products that are attached to the solid matrix by the R₁bioadhesive tag are capable of direct detection through an emission fromthe R₂ label moiety. In another exemplary embodiment, an antibody, suchas anti-dinitrophenyl (anti-DNP) for example, is attached to the solidmatrix; R₁ is an immobilization tag, such as dinitrophenyl (DNP) forexample; R₂ is a reporter or reporter precursor, such as a reactivethiol for example; and, upon silver/gold development, theoligonucleotide products that are attached to the solid matrix by the R₁tag produce a colored signal that is visible to the naked eye withoutirradiation.

[0216] Applications of the Abortive Synthesis and Detection Methods ofthe Invention

[0217] The methods of the present invention can be used in a variety ofdiagnostic contexts. For purposes of illustration, methods of assessingthe methylation state of specific genes, detecting the presence of knowngenetic mutations, detecting the presence of pathogenic organisms,detecting mRNA expression levels, and detecting and amplifying proteinsare described.

[0218] DNA Methylation

[0219] The methods of the present invention may be used in diagnosticassays which detect epigenetic changes associated with diseaseinitiation and progression by assessing the methylation state ofspecific genes and their regulatory regions that are known to beassociated with particular disease-states. DNA methylation is a cellularmechanism for altering the properties of DNA without altering the codingfunction of that sequence. The methylation reaction, which is catalyzedby DNA-(cystosine-5)-methyltransferase, involves the transfer of amethyl group from S-adenosylmethionine to the target cytosine residue toform 5-methylcytosine (5-mCyt) (FIG. 12). See Gonzalgo et al., U.S. Pat.No. 6,251,594. The areas of the genome that contain 5-mCyt at CpGdinucleotides are referred to as “CpG islands.” While changes in themethylation status of the cytosine residues in DNA CpG islands commonlyoccur in aging cells, altered gene methylation (either increased ordecreased) is frequently an early and permanent event in many types ofdisease, including cancer. CpG islands tend to be found in DNAregulatory regions that are near genes and determine whether these genesare either active or inactive. Many genes that regulate cell growth, andtherefore prevent or inhibit the development of cancer, such as tumorsuppressor genes, must be active (unmethylated) to promote normal cellgrowth. Other genes, such as oncogenes for example, must be inactive(methylated) so as not to promote abnormal cell growth.

[0220] For example, many types of cancer are associated with a distinctcombination or pattern of CpG island methylation. FIG. 16 graphicallyillustrates the manner in which altered gene methylation may beassociated with various types of cancer. The graph plots 13 exemplarycancers (prostate, kidney, bladder, esophageal, lung, gastric, colon,blood, breast, skin, brain, liver, and ovarian) against 49 genes whichhave been shown to have methylation changes that are associated with theinitiation and progression of the identified types of cancer. Each ovalin the graph (coded by cancer type) indicates an abnormal methylationstatus for a gene (ie., methylated when its normal status isunmethylated or unmethylated when its normal status is methylated).Since each type of cancer may be associated with a different pattern ofmethylation-altered genes, cancer-affected organs may potentially beidentified based upon organ-specific combinations of methylated genes.For example, in the case of prostate cancer cells, genes 4, 9, 10, 14,19, 22, 32, and 33 have been shown to exhibit abnormal methylationstates. Thus, if standardized diagnostics could easily evaluate themethylation states of these 8 genes, then the initiation, progression,and recurrence of prostate cancer could be readily monitored and moreeffective patient treatment strategies could be developed. It will beappreciated that FIG. 16 represents only a subset of the genes for whichaltered methylation states and patterns are indicative of various typesof cancer.

[0221] In an exemplary embodiment, the methods of the invention may beutilized to monitor disease initiation, progression, metastasis,recurrence, and any responses to treatment therapies by providingdiagnostic techniques, which can detect altered methylation states andpatterns. Methylated cytosine residues in a DNA fragment can be detectedbased upon the resistance of such residues to deamination by adeaminating agent, such as sodium bisulfite for example. When denatured(i.e., single-stranded) DNA is exposed to a deaminating agent, such assodium bisulfite, unmethylated cytosine (C) residues are converted intouracil residues (U), while methylated cytosine residues (5-mCyt) remainunchanged. That is, as illustrated in FIG. 14, deamination resultingfrom a treatment with sodium bisulfite causes the originallyunmethylated cytosines to change their complementary base-pairingpartner from guanine (G) to adenosine (A). However, the methylatedcytosines (5-mCyt) retain their base-pairing specificity for G. Thus,after deamination by sodium bisulfite, a target DNA sequence will haveonly as many complementary CpG islands as there were methylated CpGislands in the original, untreated target DNA sequence. Additionally, asfurther illustrated in FIG. 14, if an original, untreated target DNAsequence has no methylated CpG islands, then the bisulfite-treatedtarget DNA sequence will no longer contain any CpG islands.

[0222] In view of the foregoing, the level of methylation of the CpGislands in a target DNA sequence may be determined by measuring therelative level of unaltered CpG sites. This relative measurement may beaccomplished by initiating abortive transcription at the CpG sites thatremain after the target DNA sequence has been exposed to a deaminatingagent, such as sodium bisulfite. The sodium bisulfite reaction isperformed according to standard techniques. See, e.g., Gonzalgo et al.,U.S. Pat. No. 6,251,594. In one embodiment, as illustrated in FIG. 15, asodium bisulfite-treated DNA target sequence can be incubated with anRNA polymerase and an initiator, such as a mononucleotide initiator (5′R₁—C—OH 3′) for example. The initiator associates with the polymeraseand initiates transcription and RNA synthesis at an intact CpG site onthe DNA template. Each CpG site can direct the extension of an initiatorto synthesize an abortive transcript (e.g., 5′ R₁—CpG—R₂ 3′) through theincorporation of a suitable chain terminator (e.g., pppG—R₂), asillustrated at Sites 1, 3, and 4 in FIG. 15. Either or both of theinitiator and a chain terminating nucleotide may be modified with alabel moiety (e.g., R₁ and R₂, respectively) to allow signal detection.In an exemplary embodiment, the transcripts may be detected throughfluorescence resonance energy transfer (FRET) for example, as describedin detail above (e.g., the primer contains an energy donor (R₁) at its5′-end, and the NTP contains an energy acceptor (R₂) attached to thenucleobase).

[0223] In an alternate embodiment, a sodium bisulfite-treated DNA targetsequence may be incubated with an RNA polymerase and a dinucleotideinitiator (5′ R₁—CpG—OH 3′). The initiator then associates with thepolymerase and initiates transcription and RNA synthesis at an intactCpG site on the DNA template. Each CpG site then directs the extensionof the dinucleotide initiator to synthesize an abortive trinucleotidetranscript through the incorporation of a suitable chain terminator. Thenucleotide analog that comprises the chain terminator will depend uponthe DNA template sequence. For example, at Site 1 of FIG. 15, a suitablechain terminator would include 5′ pppA-R₂ 3′, and the resultant abortivetrinucleotide transcript would be 5′ R₁-CpGpA-R₂ 3′.

[0224] In another embodiment, as diagrammatically illustrated in FIG.15, after the target DNA sequence has been deaminated, such as bytreating the target DNA sequence with sodium bisulfite for example, atarget site probe may be used to form a bubble complex that comprises atarget CpG site on the target DNA sequence. In this embodiment, thetarget site probe is used to direct the RNA polymerase to the target CpGsite by positioning the target CpG site at the junction of asingle-stranded bubble region and a downstream duplex region on thetarget DNA sequence. In the illustrated embodiment, the target siteprobe comprises about 18-54 nucleotides: a first region which hybridizesto the target DNA sequence upstream of the target site comprises about5-20 nucleotides; an internal second region of non-base-pairednucleotides comprises about 8-14 nucleotides; and a third region whichhybridizes to the target DNA sequence downstream of the target sitecomprises about 5-20 nucleotides. The target site probe may behybridized to the target DNA sequence either before or while the DNAtarget sequence is incubated with an RNA polymerase and a suitable RNAinitiator. The polymerase associates with the RNA initiator andinitiates transcription and RNA synthesis at the CpG site on the DNAtemplate. The polymerase extends the initiator to synthesize an abortiveoligonucleotide transcript through the incorporation of a suitable chainterminator. Either or both of the initiator and a chain terminatingnucleotide may be modified with a label moiety to allow signaldetection, such as by fluorescence resonance energy transfer forexample, as described in detail above.

[0225] In another embodiment, capture probes may be designed to capturethe genes of interest, and abortive transcription initiation used todetermine the methylation status of the desired genes. For example,genes known to be associated with the progression of a particularcancer, such as colon cancer, may be monitored, including but notlimited to APC (adenomatous polyposis coli), CALCA (calcitonin), ER(estrogen receptor), GSTP1, HIC1 (hypermethylated in cancer-1), hMLH1,HPP1/TR/TENB2/TMEFF2 (Transmembrane protein with EFG-like and twofollistatin-like domains 2), LKB1/STK11. IGF2 IGF2 (Insulin-like growthfactor), MGMT (O⁶ methyl guanine methyl transferase 1), MINT25,p14(ARF), p16(INK4a)/MTSI/CDKN2A, PAX6 (paired box gene 6), RAR-Beta2,THBS1 (thrombospondin-1), Veriscan, and WT1 (Wilm's tumor suppressor).Each gene of interest could be removed from the sample by hybridizationto a capture sequence, which is unique for the gene of interest. Thecapture sequence may be immobilized on a solid matrix, including but notlimited to magnetic beads, microtiter plates, sepharose, agarose, cationexchange resins, lateral flow strips, glass beads, and microarray chips.Once the gene of interest has been removed from the sample, abortivetranscription initiation can be used to determine the methylation statusfor each particular gene.

[0226] An illustrative procedure for detecting DNA methylation statesand patterns, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isspecific for a region near a CpG island of a target gene; (b) optionallytreating the oligonucleotide capture probe with a denatured DNA samplewhich potentially contains a target DNA sequence; (c) converting anyunmethylated cytosine residues on the target DNA sequence to uracilresidues and leaving any methylated cytosine residues unaltered; (d)optionally hybridizing the target DNA sequence with a target site probe;(e) modifying at least one of an RNA initiator and nucleotidescomprising a chain terminator to enable detection of the oligonucleotidetranscript; (f) hybridizing the target DNA with the RNA initiator; and(g) extending the RNA initiator with an RNA polymerase such that the RNApolymerase reiteratively synthesizes an oligonucleotide transcript thatis complementary to a target site by incorporating complementarynucleotides comprising a chain terminator and releasing an abortiveoligonucleotide transcript without either translocating from an enzymebinding site or dissociating from the target DNA sequence; and (g)detecting and optionally quantifying the multiple abortiveoligonucleotide transcripts.

[0227] Genetic Mutations

[0228] In another aspect of the invention, the methods disclosed hereinmay be used in diagnostic assays which detect mutations in the form ofgross chromosomal rearrangements or single or multiple nucleotidealterations, substitutions, insertions, or deletions. In an exemplaryembodiment, as diagrammatically illustrated in FIG. 17, singlenucleotide polymorphisms (SNPs) may be detected through the use of anabortive oligonucleotide synthesis reaction. A known target SNP sequence(e.g., 3′ dN_(X′)pdN_(Y′)pdN_(T′)5′, where dN_(T′) is a target SNP site)can be incubated with an RNA polymerase, an RNA initiator, such as adinucleotide initiator for example, and nucleotides (e.g., a chainterminator such as 5′ pppN_(T)-R₂). The initiator binds immediatelyupstream of the target SNP sequence, associates with the polymerase, andinitiates transcription and RNA synthesis at the target SNP site. In oneembodiment, the polymerase elongates the initiator by incorporating thechain terminator to produce an abortive trinucleotide product. Either orboth of the initiator and a chain terminating nucleotide may be modifiedwith a label moiety (R₁ and R₂, respectively) to allow signal detection.In an exemplary embodiment, the transcripts may be detected throughfluorescence resonance energy transfer (FRET) for example, as describedin detail above (e.g., the initiator contains an energy donor (R₁) atits 5′-end, and the chain terminator contains an energy acceptor (R₂)attached to the nucleobase).

[0229] An illustrative procedure for detecting mutations in a target DNAsequence (FIG. 18), therefore, may include the following process steps:(a) optionally immobilizing a capture probe designed to hybridize with atarget DNA sequence which includes a mutation; (b) optionallyhybridizing the capture probe with a DNA sample which potentiallycontains the target DNA sequence; (c) optionally hybridizing the targetDNA sequence with a target site probe; (d) modifying at least one of anRNA initiator (R₁N_(I)—OH) and nucleotides comprising a chain terminator(pppN_(T)-R₂)to enable detection of the oligonucleotide transcriptsynthesized by the RNA polymerase; (e) hybridizing the target DNAsequence with the RNA initiator; (f) extending the RNA initiator with anRNA polymerase such that the RNA polymerase reiteratively synthesizes anoligonucleotide transcript that is complementary to a target mutationsite by incorporating complementary nucleotides comprising a chainterminator and releasing an abortive oligonucleotide transcript withouteither translocating from an enzyme binding site or dissociating fromthe target DNA sequence; and (g) detecting and optionally quantifyingthe multiple abortive oligonucleotide transcripts.

[0230] Pathogenic Organisms

[0231] In another aspect of the invention, the methods disclosed hereinmay be used in diagnostic assays which detect the presence of aparticular nucleic acid (DNA or RNA), thereby serving to indicate thepresence of either a particular or a generic organism which contains thegene, or which permit genetic typing of a particular organism withoutthe need for culturing the organism. The test sample may be suspected ofcontaining a target nucleic acid sequence from a particularmicroorganism, such as bacteria, yeast, viruses, viroids, molds, fungi,and the like. The test sample may collected from a variety of sourcesincluding but not limited to, animal, plant or human tissue, blood,saliva, semen, urine, sera, cerebral or spinal fluid, pleural fluid,lymph, sputum, fluid from breast lavage, mucusoal secretions, animalsolids, stool, cultures of microorganisms, liquid and solid food andfeedproducts, waste, cosmetics, air, and water. In another aspect of theinvention, the methods disclosed herein may be used in diagnostic assayswhich detect the presence of a particular nucleic acid (DNA or RNA),thereby serving to indicate the presence of either a particular or ageneric pathogenic organism which contains the gene, or which permitgenetic typing of a particular organism without the need for culturingthe organism. In an exemplary embodiment, as diagrammaticallyillustrated in FIG. 19, an oligonucleotide capture probe that issequence-specific for a target pathogen polynucleotide is attached to asolid matrix, such as a microtiter plate for example, and the captureprobe is treated under hybridizing conditions with a test sample whichpotentially contains the target pathogen polynucleotide. The test samplemay be suspected of containing a target nucleic acid sequence from aparticular pathogen, such as, for example, a microorganism, such asbacteria, yeast, viruses, viroids, molds, fungi, and the like. The testsample may collected from a variety of sources including but not limitedto, animal, plant or human tissue, blood, saliva, semen, urine, sera,cerebral or spinal fluid, pleural fluid, lymph, sputum, fluid frombreast lavage, mucusoal secretions, animal solids, stool, cultures ofmicroorganisms, liquid and solid food and feedproducts, waste,cosmetics, air, and water.

[0232] The target pathogen polynucleotide may be either RNA or DNA. Atarget pathogen polynucleotide that is present in the test samplehybridizes to the capture probe, and a washing step is then performed toremove any components of the test sample that were not immobilized bythe capture probe. Target DNA or RNA may be retrieved by addition ofspecific sequences via primer extension, for example. In an exemplaryembodiment, the captured target pathogen polynucleotide is hybridizedwith an abortive promoter cassette (APC). The APC linker sequenceincludes a single-stranded overhang region on either its 3′ or 5′ end(depending upon the orientation needed to create an antiparallel hybridwith the capture probe). In other words, the APC linker is complementaryto the sequence on the free end of the captured target pathogenpolynucleotide, thereby permitting the APC linker to hybridize to thetarget pathogen polynucleotide.

[0233] An initiator and a polymerase are added to the reaction mixture.The initiator hybridizes within the bubble region of the APC at aposition that facilitates catalysis of a synthesis reaction by asuitable polymerase at the target site. The initiator may be RNA or DNA,may comprise from about 1 to 25 nucleotides, and may include one or morenucleotide analogs as well as nucleotides. The polymerase may be anRNA-dependent or DNA-dependent RNA polymerase. The DNA or RNA APC may ormay not be attached to other molecules, such as proteins, for example.In an exemplary embodiment, the APC comprises DNA, the initiator is RNA,and the polymerase is a DNA-dependent RNA polymerase.

[0234] During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotidesthat have been added to the reaction mixture. As the polymerase reactionproceeds, the polymerase extends the initiator, as directed by the APCtemplate sequence within the bubble region, by incorporatingcomplementary nucleotides, including a suitable chain terminator, thatare present in the reaction mixture. When the polymerase incorporates achain terminator into the nascent oligonucleotide product, chainelongation terminates due to the polymerase's inability to catalyze theaddition of a nucleotide at the 3′ position on the pentose ring of theincorporated chain terminator. Consequently, the polymerase aborts theinitiated synthesis event by releasing the oligonucleotide product andreinitiating the synthesis reaction at the target site. Either or bothof the initiator and a chain terminating nucleotide may be modified witha label moiety to allow signal detection. In an exemplary embodiment,the oligonucleotide products may be detected through fluorescenceresonance energy transfer (FRET), as described above (e.g., theinitiator contains an energy donor (R₁) at its 5′-end, and the chainterminator contains an energy acceptor (R₂) attached to the nucleobase).

[0235] An illustrative procedure for detecting the presence of pathogens(FIG. 20), therefore, may include the following process steps: (a)optionally immobilizing a capture probe designed to hybridize with atarget pathogen polynucleotide; (b) optionally hybridizing the captureprobe with a test sample which potentially contains a target pathogenpolynucleotide. The target nucleic acid may be copied to DNA via reversetranscription (for RNA pathogens) or primer extension (for DNApathogens). In both bases, a DNA sequence corresponding to the AbortivePromoter Cassette (APC) linker will be added to the target copy (FIG.1); (c) optionally washing the captured target pathogen polynucleotideto remove any unhybridized components of the test sample; (d)hybridizing the captured target pathogen polynucleotide with an abortivepromoter cassette; (e) modifying at least one of a initiator andnucleotides comprising a chain terminator to enable detection of theoligonucleotide product synthesized by the polymerase; (f) hybridizingthe abortive promoter cassette with a initiator; (g) extending theinitiator with a polymerase such that the polymerase reiterativelysynthesizes an oligonucleotide product that is complementary to a targetsite by incorporating complementary nucleotides comprising a chainterminator and releasing an abortive oligonucleotide product withouteither translocating from an enzyme binding site or dissociating fromthe APC; and (h) detecting and optionally quantifying the multipleabortive oligonucleotide products.

[0236] The present invention is useful for detecting pathogens inmammals. In particular the invention is useful for the detection ofbacteria, viruses, fungus, molds, amoebas, prokaryotes, and eukaryotes.Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses,rabbits and humans. Particularly preferred are humans.

[0237] The methods of the invention are particularly useful formonitoring the presence or absence of pathogenic nucleic acids andproteins. The invention can be used to detect, diagnose, and monitordiseases, and/or disorders associated with pathogenic polypeptides orpolynucleotides. The invention provides for the detection of theaberrant expression of a polypeptide or polynucleotide. The methodcomprises (a) assaying the expression of the polypeptide orpolynucleotide of interest in cells, tissue or body fluid of anindividual using the methods of abortive initiation transcriptiondescribed above, and (b) comparing the level of gene expression, proteinexpression, or presence of sequences of interest with a standard gene orprotein expression level or seqeunce of interest, whereby an increase ordecrease in the assayed polypeptide or polynucleotide level compared tothe standard level is indicative of aberrant expression indicatingpresence of a pathogen of interest..

[0238] The presence of an abnormal amount of transcript in biopsiedtissue or body fluid from an individual may provide a means fordetecting the disease prior to the appearance of actual clinicalsymptoms. A more definitive diagnosis of this type may allow healthprofessionals to employ preventative measures or aggressive treatmentearlier thereby preventing the development or further progression of thedisease caused by the pathogen.

[0239] The invention is particularly useful for monitoring the presenceof pathogenic organisms including but not limited to E. coli,Steptococcus, Bacillus, Mycobacterium, HIV, and Hepatitis.

[0240] The methods of the invention may be used to test for pathogenicmicroorganisms in aqueous fluids, in particular water (such as drinkingwater or swimming or bathing water), or other aqueous solutions (such asfermentation broths and solutions used in cell culture), or gases andmixtures of gases such as breathable air, and gases used to sparge,purge, or remove particulate matter from surfaces. Breathable air fromany source including but not limited to homes, schools, classrooms,workplaces, aircraft, spacecraft, cars, trains, buses, and any otherbuilding or structure where people gather, may be tested for thepresence of pathogenic microorganisms.

[0241] mRNA Expression

[0242] In another aspect of the invention, the methods disclosed hereinmay be used in diagnostic assays which detect messenger RNA (mRNA)expression levels in a quantitative or non-quantitative manner. In anexemplary embodiment, as diagrammatically illustrated in FIG. 21, anoligonucleotide capture probe that is sequence-specific for a targetmRNA sequence is attached to a solid matrix, such as a microtiter platefor example, and the capture probe is treated under hybridizingconditions with a test sample which is suspected of containing thetarget mRNA sequence. A target mRNA sequence that is present in the testsample hybridizes to the capture probe, and a washing step is thenperformed to remove any components of the test sample that were notimmobilized by the capture probe. The captured target mRNA sequence isthen hybridized with an abortive promoter cassette (APC). In theillustrated embodiment, the APC has an APC linker sequence whichincludes a single-stranded poly-T overhang on its 3′ end that iscomplementary to the poly-A tail on the 3′ end of the target mRNAsequence, thereby permitting the APC linker to hybridize to the poly-Atail of the target mRNA.

[0243] An initiator and a polymerase are added to the reaction mixture.The initiator hybridizes within the bubble region of the APC, upstreamof the target site, and facilitates catalysis of a synthesis reaction bya suitable polymerase at the target site. The initiator may comprisefrom about 1 to 25 nucleotides, and may include one or more nucleotideanalogs as well as nucleotides. The polymerase may be an RNA-dependentor DNA-dependent RNA polymerase. The APC may or may not be attached toother molecules, such as proteins, for example. In an exemplaryembodiment, the APC comprises DNA, the initiator is RNA, and thepolymerase is a DNA-dependent RNA polymerase.

[0244] During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotideswhich have been added to the reaction mixture. As the polymerasereaction proceeds, the polymerase extends the initiator, as directed bythe APC template sequence within the bubble region, by incorporatingcomplementary nucleotides, including a chain terminator, that arepresent in the reaction mixture. When the polymerase incorporates achain terminator into the nascent oligonucleotide product, chainelongation terminates due to the polymerase's inability to catalyze theaddition of a nucleotide at the 3′ position on the pentose ring of theincorporated chain terminator. Consequently, the polymerase aborts theinitiated synthesis event by releasing the oligonucleotide product andreinitiating the synthesis reaction at the target site. Either or bothof the initiator and a chain terminating nucleotide may be modified witha label moiety to allow signal detection, such as by fluorescenceresonance energy transfer for example, as described in detail above.

[0245] An illustrative procedure for detecting mRNA expression levels,therefore, may include the following process steps: (a) optionallyimmobilizing a capture probe designed to hybridize with a specific orgeneral mRNA sequence; (b) optionally hybridizing the capture probe witha test sample which potentially contains a target mRNA sequence; (c)optionally washing the captured target mRNA sequence to remove anyunhybridized components of the test sample; (d) hybridizing the capturedtarget mRNA sequence with an abortive promoter cassette; (e) modifyingat least one of a initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide productsynthesized by the polymerase; (f) hybridizing the abortive promotercassette with the initiator; (g) extending the initiator with apolymerase such that the polymerase reiteratively synthesizes anoligonucleotide product that is complementary to a target site byincorporating complementary nucleotides comprising a chain terminatorand releasing an abortive oligonucleotide product without eithertranslocating from an enzyme binding site or dissociating from the APC;and (h) detecting and optionally quantifying the multiple abortiveoligonucleotide products.

[0246] Protein Detection

[0247] In another aspect of the invention, the methods disclosed hereinmay be used in diagnostic assays which detect proteins. As shown in FIG.22, an abortive promoter cassette linker can be made with a proteinmodifier group attached, such that the linker is complementary to theAPC linker attached to the APC.

[0248] An illustrative procedure for detecting proteins, therefore, mayinclude the following process steps: (a) attaching a short piece of DNAof a defined sequence (APC linker) to a protein via a primary amine, asecondary amine, or a sulfhydral group; (b) retrieving and immobilizingthe modified protein with an antibody or some other affinity agentagainst the protein; and (c) attaching an abortive promoter cassette tothe protein by hybridization of the APC cassette to the APC linker onthe labeled protein; (d) detecting the protein by (i) treating the DNAwith an initiator nucleotide under hybridizing conditions; and (ii)treating the DNA with an RNA polymerase and nucleotides or nucleotideanalogs that permit detection. Process (d) occurs repeatedly for eachRNA polymerase bound.

[0249] Cancer Detection

[0250] The present invention is useful for detecting cancer in mammals.In particular the invention is useful during diagnosis of cancer.Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses,rabbits and humans. Particularly preferred are humans.

[0251] The methods of the invention are particularly useful formonitoring the status of DNA methylation, genetic mutations, mRNAexpression patterns, and protein expression patterns. The invention canbe used to detect, diagnose, and monitor diseases, and/or disordersassociated with the aberrant expression and/or activity of a polypeptideor polynucleotide. The invention provides for the detection of theaberrant expression of a polypeptide or polynucleotide, the presence ofmutations, and changes in methylation status of DNA. The methodcomprises (a) assaying the expression of the polypeptide orpolynucleotide of interest in cells, tissue or body fluid of anindividual using the methods of abortive initiaton transcriptiondescribed above, and (b) comparing the level of gene expression, proteinexpression, or presence of sequences of interest with a standard geneexpression level, whereby an increase or decrease in the assayedpolypeptide or polynucleotide level compared to the standard level isindicative of aberrant expression indicating presence of cancer or apathogen of interest..

[0252] The presence of an abnormal amount of transcript in biopsiedtissue or body fluid from an individual may indicate a predispositionfor the development of cancer or a disease of interest, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer or disease caused by the pathogen.

[0253] The diagnostic assays of the invention can be used for thediagnosis and prognosis of any disease, including but not limited toAlzheimer disease, muscular dystrophy, cancer, breast cancer, coloncancer, cystic fibrosis, fragile X syndrome, hemophilia A and B, Kennedydisease, ovarian cancer, lung cancer, prostate cancer, retinoblastoma,myotonic dystrophy, Tay Sachs disease, Wilson disease, and Williamsdisease. These assays are believed to be particularly useful for thediagnosis and prognosis of all types of cancer.

[0254] Kits of the Invention

[0255] The invention also provides kits for carrying out the methods ofthe invention. Such kits comprise, in one or more containers, usuallyconveniently packaged to facilitate their use in assays, quantities ofvarious compositions essential for carrying out the assays in accordancewith the invention. Thus, the kits comprise one or more initiatorsaccording to the invention. The kits may additionally comprise an enzymewith polymerase activity, such as an RNA and/or DNA polymerase forexample, to extend the primer of the kit, as well as reagents forprocessing a target nucleic acid. The kit may also comprise nucleotidesand/or nucleotide analogs to enable detection of the oligonucleotideproducts synthesized by the methods of the invention. The kits may alsoinclude oligonucleotide target site probes for forming a bubble complexon the target nucleic acid. The kit may also contain an abortivepromoter cassette. The kits may also contain components for thecollection and transport of materials, including but not limited to,membranes, affinity materials, test tubes, petri dishes, and dipsticks.The kit may also include microtiter plates, bio-chips, magnetic beads,gel matrices, or other forms of solid matrices to which anoligonucleotide capture probe, which is specific for a particular targetsequence, has been bound. The relative amounts of the components in thekits can be varied to provide for reagent concentrations thatsubstantially optimize the reactions involved in the practice of themethods disclosed herein and/or to further optimize the sensitivity ofany assay.

[0256] The test kits of the invention can also include, as is well-knownto those skilled in the art, various controls and standards, such assolutions of known target nucleic acid concentration, including notarget sequence (negative control), to ensure the reliability andaccuracy of the assays carried out using the kits and to permitquantitative analyses of test samples using the kits. Optionally, thekits may include a set of instructions, which are generally writteninstructions, though the instructions may be stored on electronicstorage media (e.g., magnetic diskette or optical disk), relating to theuse of the components of the methods of the invention. The instructionsprovided with the kit generally also include information regardingreagents (whether included or not in the kit) necessary or preferred forpracticing the methods of the invention, instructions on how to use thekit, and/or appropriate reaction conditions.

EXAMPLES

[0257] The following examples are provided for purposes of illustrationonly and not of limitation. Those of skill in the art will readilyrecognize a variety of non-critical parameters which could be changed ormodified to yield essentially similar results.

Example 1 Synthesis of a Dye Labeled Initiator

[0258] One of several reactions to chemically modify a nucleotide isdescribed herein. 5′ a-S-CTP, which was purchased from TriLinkBioTechnologies, was treated following the manufacturer's instructionswith calf intestinal phosphatase. The phosphatase treatment is importantbecause it increases the efficiency of the labeling reaction. Followingphosphatase treatment, 12.5 mM α-S-CMP was mixed with 5 μl of 0.2 MNaHCO3, 15 μl of DMF, and 15 μl of 90 mM IAEDANS (purchased fromMolecular Probes) in DMF, and incubated at room temperature for 1 hour.The reaction was extracted with 5 volumes water saturated ethyl ether.The aqueous phase was removed and the ether eliminated by evaporation.Thin layer chromatography was performed following standard protocolsknown in the art, and demonstrated that the reaction successfullyproduced 5′-IEADANS-S-CMP (FIG. 26).

Example 2 RNA Primer-Initiated Abortive Transcription with an RNAPolymerase

[0259] Reaction conditions have been optimized for abortive trancriptioninitiaton. The components and concentrations of Buffer T favor abortivetranscription initiation. Buffer T is comprised of: 20 mM Tris-HCl pH7.9, 5 mM MgCl₂, 5 mM beta-mercaptoethanol, 2.8% (v/v) glycerol. Primersare either ribonucleoside-triphosphates (NTPs) or dinucleotides rangingin concentration from 0.2-1.3 mM. Final NTP concentrations range from0.2-1.3 mM. The high ends of the concentration ranges are designed forpreparative abortive transcription. The template DNA concentration isless than 2 μM in terms of phosphate. E. coli RNA polymerase is added toa final concentration of between 15 nM and 400 nM. Either holoenzyme orcore can be used with a single-stranded template DNA. Yeast inorganicpyrophosphatase is added to 1 unit/ml in preparative reactions toprevent the accumulation of pyrophosphate. At high concentrationspyrophosphate can reverse the synthesis reaction causing RNA polymeraseto regenerate NTPs at the expense of the RNA products. One unit ofpyrophosphatase is defined as the amount of enzyme to liberate 1.0 μM ofinorganic orthophosphate per min. at 25° C. and pH 7.2. Reactions areincubated at 37° C. for up to 72 hours for preparative reactions. Theseconditions are representative; for specific templates, optimization ofparticular components and concentrations may enhance the efficiency ofabortive initiation.

[0260] Three different initiators were used in this example: (1)TAMARA-ApG; (2) Biotin ApG; and (3) ApG. The target nucleic acidtemplate was denatured by boiling for 5 minutes at 95° C. andimmediately placing on ice. Each reaction was prepared as follows:

[0261] 5.0 μl 1×Buffer T

[0262] 2.5 μl of a-32P-UTP

[0263] 14.3 μl ddH2O

[0264] 1 μl of E. coli RNA polymerase (1 U/μl)

[0265] 100 ng (2 μl) of template DNA

[0266] 10 nmoles (1.2 μl) of initiator

[0267] 22.8 μl of reaction buffer

[0268] Incubate at 37° C. for 12-16 hours. Thin layer chromatography wasperformed using standard methods known in the art to determine theextent of incorporation of UTP in the third position (FIG. 27).

[0269] Both TAMARA-ApG and biotin ApG allowed for incorporation of thenucleotide UTP. Biotin ApG incorporated more efficiently thanTAMARA-ApG, but not as efficient as ApG.

Example 3 Abortive Initiation Reaction with a Labeled Terminator

[0270] Abortive transcription initiation reactions may be performed witha labeled initiator and/or a labeled terminator. The following reactionconditions were used to incorporate a labeled terminator:

[0271] 5 μl 1×Buffer T

[0272] 3 μl 100 ng denatured DNA template (pBR322)

[0273] 13.5 μl dd H₂O

[0274] 1 μl E. coli RNA polymerase

[0275] 1.2 μl dinucleotide initiator ApG

[0276] 1.5 μl of 7 mM SF-UTP

[0277] Incubate mixture at 37° C. for 16 hours in temperature controlledmicrotitre plate reader. Thin layer chromatography was performed usingstandard methods known in the art, and demonstrated that the labeledtrinucleotide ApGpU was generated (FIG. 28).

Example 4 Fluorescent Energy Transfer Between Donors and Acceptors

[0278] The above examples have demonstrated that both labeled initiatorsand terminators can be incorporated into the oligonucleotide products.One efficient method to measure incorporation of the labeled nucleotidesis by Fluorescent Resonance Energy Transfer. The following conditionswere used to demonstrate FRET between a labeled initiator and a labeledterminator:

[0279] 5 μl 1×Reaction Buffer (Buffer T)

[0280] 3 μl denatured DNA template (300 ng pBR322)

[0281] 13.5 μl dd H₂O

[0282] 1 μl E. coli RNA polymerase

[0283] 1.2 μl Initiator (TAMARA-ApG or ApG or Biotin-ApG)

[0284] 1.5 μl of of 7 mM SF-UTP

[0285] The reaction mixture was incubated at 37 C. for 16 hours intemperature controlled microtitre plate reader, which was set to read atthe following parameters: Ex 485, Em 620, Gain 35, 99 reads/well/cycle.Under the reaction conditions described above, the RNA polymerasereiteratively synthesizes an oligonucleotide product composed of theinitiator (TAMARA-SpApG) and the terminator (SF-UTP).

[0286] Formation of the oligonucleotide product, TAMARA-SpApGpU-SF,places the initiator and the terminator within 80 angstroms of eachother, which allows for the transfer of energy between the chemicalmoieties. Energy is transferred from the donor, which is SF-UTP, to theacceptor, which is TAMARA-ApG. This transfer of energy can be detectedand/or quantitated by a change in wavelength emission of TAMARA (TAMARAAbosrbance 540 nm; Emission=565 nm)

[0287] As shown in FIG. 29A, as the oligonucleotide product isgenerated, energy transfer occurs between TAMARA-SpApG and SF-UTP, whichchanges the wavelength at which TAMARA emits. If RNA polymerase or DNAis omitted from the reaction, there is no transfer of energy between theinitiator and the terminator, and no change in the wavelength at whichTAMARA emits (FIGS. 29B and 29C).

Example 5 Determination of the Methylation Status of Specific Residuesof the CDKN2A gene

[0288] The sample to be analyzed is collected from a human stool sample.Methods of DNA extraction from stool samples are well known in the art,and commercial kits are avialalbe for extracting human DNA from stoolsamples, such as QIAamp DNA Stool Mini Kit from Qiagen (Valencia,Calif.).

[0289] After extraction, the sample is applied to the wells of amicrotiter plate, which contain a capture probe for the gene ofinterest, in this particular example, the capture probe is for CDKN2Agene. The nucleotide sequence of a representative capture probe for theCpG islands of the CDKN2A gene is as follows:

[0290] ATATACTGGGTCTACAAGGTTTAAGTCAACCAGGGATTGAAATATAACTTTTAAACAGAGCTGG. The DNA sample is incubated with the capture probe toallow hybridization. A representative hybridization protocol is asfollows: (1) prehybridize with 2.5×SSC, 5×Denhardts at room temperaturefor 30 minutes; (2) hybridize with 2.5×SSC, 5×Denhardts, 30% formamideat room temperature for 2 hours; (3) wash twice with 1×SSC at 42° C. for10 minutes, maintaining 42° C.; and (4) wash three times with 0.1×SSC at42° C. for 10 minutes, maintaining 42° C.

[0291] The DNA is treated with a deaminating agent, such as sodiumbisulfite, which will de-aminate the unmethylated C's in the DNA, whileleaving the methylated C's unaltered. The wells are then washed undermedium stringency conditions to remove the remaining sodium bisulfite.

[0292] A representative transcription reaction is comprised of thefollowing components: E. coli holoenzyme RNA polymerase; reactionbuffer: 10 nM Tris-HCl, pH 7.0; 10 mM KCl; 0.5 mM Na₂EDTA; and 50 mg/mlBSA; an initiator, and nucleotide analogs. The reaction conditions forparticular nucleotide sequence may vary. Other polymerases may be used,such as E. coli T7, or SP6. The reaction buffer can be optimized toincrease abortive initiation events by adjusting the salt concentration,divalent cations and concentrations, the glycerol content, and theamount and type of reducing agent to be used.

[0293] The initiator will be a 5′-αSpCpG dimer labeled through the 5′-Swith fluorescein, which fucntions as the donor in this reaction. Thenucleotide analog(s) will be labeled with TAMARA, which will function asthe acceptor in this reaction. The initiator can be labeled with eitherthe donor or the acceptor in the FRET reaction, and dependending uponthe fluorescent molecule used to label the initiator, the nucleotideanalog(s) will be labeled with either a donor or an acceptor.

[0294] Fluorescein is excited using a 360 nm wavelength filter; theresulting emission peak is at about 515 nm. If the TAMARA is in closeproximity to the fluorescein, it becomes excited at 542 nm, resulting inan emission peak of 568 nm. The near ultraviolet wavelength excties thefluorescein but not the rhodamine. Therefore signal will only begenerated if the fluorescein is in close proximity to the rhodamine.This signal can be generated and monitored in a fluorescent microtitreplate reader that has been fitted with specific excitation and emissionfilters for this FRET pair. These filters and plate readers arecommercially available from a number of sources, although most clinicallabs and research facilities already use a fluorescent microtitre platereader.

[0295] In the foregoing specification, the invention has been describedwith reference to specific embodiments. However, it will be appreciatedthat various modifications and changes can be made without departingfrom the scope of the present invention as set forth in the claimsbelow. The specification and figures are to be regarded in anillustrative manner, rather than a restrictive one, and all suchmodifications are intended to be included within the scope of presentinvention. Accordingly, the scope of the invention should be determinedby the appended claims and their legal equivalents, rather than by theexamples given above. For example, the steps recited in any of themethod or process claims may be executed in any order and are notlimited to the order presented in the claims.

What is claimed is:
 1. A method for detecting multiple reiteratedoligonucleotides from a target DNA or RNA polynucleotide, said methodcomprising: (a) hybridizing an initiator with a single stranded targetpolynucleotide (b) incubating said target polynucleotide and initiatorwith an RNA-polymerase, and a terminator; (c) synthesizing multipleoligonucleotides from said target polynucleotide, wherein said initiatoris extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple reiterativeoligonucleotides; and (d) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts of a polynucleotide of interest.2. The method of claim 1, further comprising detecting or quantifyingsaid reiteratively synthesized oligonucleotide by modifying a nucleosideor nucleotide in at least one of the members selected from the groupconsisting of said terminator, and said initiator.
 3. The method ofclaim 2, wherein said modifying comprises incorporating a label moiety.4. The method of claim 3, wherein said label moiety comprises afluorophore moiety.
 5. The method of claim 4, wherein said fluorophoremoiety comprises a fluorescent energy donor and a fluorescent energyacceptor wherein said moiety is detected or quantified by fluorescenceresonance energy transfer.
 6. The method of claim 1, wherein saidpolymerase is selected from the group consisting of: a DNA-dependent RNApolymerase, an RNA-dependent RNA polymerase and a modifiedRNA-polymerase, and a primase.
 7. The method of claim 6, wherein saidpolymerase comprises an RNA polymerase derived from one of E. coli, E.coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimuriumbacteriophage SP6.
 8. The method of claim 1, wherein said initiator isan RNA primer.
 9. The method of claim 1, wherein said initiatorcomprises a molecule selected from the group consisting of: nucleosides,nucleoside analogs, 1-25 nucleotides, 26-50 nucleotides, 51-75nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225nucleotides, 226-250 nucleotides, greater than 250 nucleotides, andnucleotide analogs.
 10. The method of claim 1, wherein said abortiveoligonucleotides being synthesized are one of the lengths selected fromthe group consisting of: about 2 to about 26 nucleotides, about 26 toabout 50 nucleotides, and about 50 nucleotides to about 100 nucleotides,and greater than 100 nucleotides.
 11. The method of claim 1, whereinsaid incubating further comprises a target site probe specific for aregion on said single-stranded target polynucleotide.
 12. The method ofclaim 1, wherein said chain terminator comprises a nucleotide analog.13. A method of detecting multiple reiterated oligonucleotides from atarget DNA or RNA polynucleotide, said method comprising: (a)hybridizing an initiator to a single-stranded target polynucleotide; (b)incubating said target polynucleotide and initiator with a target siteprobe, an RNA-polymerase, and a terminator, wherein said target siteprobe hybridizes with said target polynucleotide; (c) synthesizing anoligonucleotide transcript that is complementary to said target sitefrom said target polynucleotide, wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotidetranscript, thereby synthesizing multiple reiterative oligonucleotidetranscripts; and (d) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts.
 14. The method of claim 13,wherein said target site probe size is selected from the groupconsisting of: about 20 to about 50 nucleotides, about 51 to about 75nucleotides, about 76 to about 100 nucleotides and greater than 100nucleotides.
 15. The method of claim 13, further comprising detecting orquantifying said reiteratively synthesized oligonucleotide by modifyinga nucleotide in at least one of the members selected from the groupconsisting of said terminator, said initiator, and said target-siteprobe.
 16. The method of claim 15, wherein said modifying comprisesincorporating a label moiety.
 17. The method of claim 16, wherein saidlabel moiety comprises a fluorophore moiety.
 18. The method of claim 17,wherein said fluorophore moiety comprises a fluorescent energy donor anda fluorescent energy acceptor wherein said moiety is detected orquantified by fluorescence resonance energy transfer.
 19. The method ofclaim 13, wherein said polymerase is selected from the group consistingof: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase anda modified RNA polymerase, and a primase.
 20. The method of claim 19,wherein said polymerase comprises an RNA polymerase derived from one ofE. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S.typhimurium bacteriophage SP6.
 21. The method of claim 13, wherein saidinitiator is an RNA primer.
 22. The method of claim 13, wherein saidinitiator comprises nucleotides selected from the group consisting of:1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250nucleotides, and greater than 250 nucleotides
 23. The method of claim13, wherein said abortive oligonucleotides being synthesized are one ofthe lengths selected from the group consisting of: about 2 to about 26nucleotides, about 26 to about 50 nucleotides and about 50 nucleotidesto about 100 nucleotides, and greater than 100 nucleotides.
 24. Themethod of claim 13, wherein said mixture in “a” further comprises atarget site probe specific for a region on said single-stranded targetpolynucleotide.
 25. The method of claim 13, wherein said chainterminator comprises a nucleotide analog.
 26. A method for detectingmethylated cytosine residues at CpG sites in a target polynucleotide,comprising: (a) deaminating a single-stranded target DNA sequence underconditions which convert unmethylated cytosine residues to uracilresidues while not converting methylated cytosine residues to uracil;(b) hybridizing an initiator with a single stranded targetpolynucleotide; (c) incubating said deaminated target polynucleotide andsaid initiator with a terminator, and an RNA-polymerase, wherein atleast one of said initiator, or terminator is modified to enabledetection of the CG sites; (d) synthesizing an oligonucleotidetranscript that is complementary to said CG sites from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotide transcript therebysynthesizing multiple reiterative oligonucleotide transcripts; and (e)detecting or quantifying said reiteratively synthesized oligonucleotidetranscripts.
 27. A method for detecting methylated cytosine residues ata CpG site in a target gene, said method comprising: (a) deaminating asingle-stranded target DNA polynucleotide under conditions which convertunmethylated cytosine residues to uracil residues while not convertingmethylated cytosine residues to uracil; (b) hybridizing a target siteprobe with said single stranded target polynucleotide, (c) incubatingsaid target polynucleotide and target site probe with, an initiator, aterminator, and an RNA-polymerase, wherein at least one of saidinitiator, or said terminator are complementary to the CpG site; (d)synthesizing an oligonucleotide transcript that is complementary to saidtarget site from said target polynucleotide, wherein said initiator isextended until said terminator is incorporated into saidoligonucleotides, thereby synthesizing multiple reiterativeoligonucleotide transcripts; and (e) detecting or quantifying saidreiteratively synthesized oligonucleotide transcripts.
 28. The method ofclaim 27, wherein said target site probe size is selected from the groupconsisting of: about 5-19; about 20 to about 50 nucleotides, about 51 toabout 75 nucleotides, about 76 to about 100 nucleotides and greater than100 nucleotides.
 29. The method of claim 27, further comprisingdetecting or quantifying said reiteratively synthesized oligonucleotideby modifying a nucleotide in at least one of the members selected fromthe group consisting of said terminator, and said initiator.
 30. Themethod of claim 29, wherein said modifying comprises incorporating alabel moiety.
 31. The method of claim 30, wherein said label moietycomprises a fluorophore moiety.
 32. The method of claim 31, wherein saidfluorophore moiety comprises one of a fluorescent energy donor and afluorescent energy acceptor wherein said moiety is detected orquantified by fluorescence resonance energy transfer.
 33. The method ofclaim 27, wherein said polymerase is selected from the group consistingof: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase anda modified RNA polymerase, and a primase.
 34. The method of claim 27,wherein said polymerase comprises an RNA polymerase derived from one ofE. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S.typhimurium bacteriophage SP6.
 35. The method of claim 27, wherein saidinitiator is an RNA primer.
 36. The method of claim 27, wherein saidinitiator comprises nucleotides selected from the group consisting of:1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250nucleotides, and greater than 250 nucleotides
 37. The method of claim27, wherein said abortive oligonucleotides being synthesized are one ofthe lengths selected from the group consisting of: about 2 to about 26nucleotides, about 26 to about 50 nucleotides and about 50 nucleotidesto about 100 nucleotides, and greater than 100 nucleotides.
 38. Themethod of claim 27, wherein said chain terminator comprises a nucleotideanalog.
 39. The method of claim 26 or 27, wherein deaminating asingle-stranded target DNA sequence comprises treating saidsingle-stranded target DNA sequence with sodium bisulfite.
 40. Themethod of claim 27, wherein said target site probe and said target DNAsequence form a bubble complex comprising a first double-stranded regionupstream of said target CpG site, a single-stranded region comprisingsaid target CpG site, and a second double-stranded region downstream ofsaid target CpG site.
 41. A method for detecting the presence or absenceof mutations in a target DNA sequence, the method comprising (a)hybridizing a target site probe to a single-stranded DNA polynucleotide,wherein said DNA polynucleotide may contain a mutation relative to anormal or wild type gene; (b) incubating said target polynucleotide andtarget-site probe with an RNA-polymerase, an initiator, and aterminator; (c) synthesizing an oligonucleotide transcript from saidtarget polynucleotide that is complementary to a target mutation site,wherein said initiator is extended until said terminator is incorporatedinto said oligonucleotides thereby synthesizing multiple abortivereiterative oligonucleotides; and (d) determining the presence orabsence of a mutation by detecting or quantifying said reiterativelysynthesized oligonucleotides transcribed from said target DNApolynucleotide.
 42. The method of claim 41, wherein said target siteprobe size is selected from the group consisting of: about 20 to about50 nucleotides, about 51 to about 75 nucleotides, about 76 to about 100nucleotides and greater than 100 nucleotides.
 43. The method of claim41, further comprising detecting or quantifying said reiterativelysynthesized oligonucleotide by modifying a nucleotide in at least one ofthe members selected from the group consisting of said terminator, andsaid initiator.
 44. The method of claim 43, wherein said modifyingcomprises incorporating a label moiety.
 45. The method of claim 44,wherein said label moiety comprises a fluorophore moiety.
 46. The methodof claim 45, wherein said fluorophore moiety comprises a fluorescentenergy donor and a fluorescent energy acceptor wherein said moiety isdetected or quantified by fluorescence resonance energy transfer. 47.The method of claim 41, wherein said polymerase is selected from thegroup consisting of: a DNA-dependent RNA polymerase, an RNA-dependentRNA polymerase and a modified RNA polymerase, and a primase.
 48. Themethod of claim 47, wherein said polymerase comprises an RNA polymerasederived from one of E. coli, E. coli bacteriophage T7, E. colibacteriophage T3, and S. typhimurium bacteriophage SP6.
 49. The methodof claim 41, wherein said abortive oligonucleotides being synthesizedare one of the lengths selected from the group consisting of: about 2 toabout 26 nucleotides, about 26 to about 50 nucleotides and about 50nucleotides to about 100 nucleotides, and greater than 100 nucleotides.50. The method of claim 41, wherein said chain terminator comprises anucleotide analog.
 51. The method of claim 41, wherein said mutation isselected from the group consisting of: a deletion, an insertion, asubstitution, a chromosomal rearrangement, and a single nucleotidepolymorphism.
 52. The method of claim 41, wherein said initiatorcomprises nucleotides selected from the group consisting of: 1-25nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides,101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides,176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, andgreater than 250 nucleotides
 53. The method of claim 41, wherein saidtarget site probe and said target DNA sequence form a bubble complexcomprising a first double-stranded region upstream of said targetmutation site, a single-stranded region comprising said target mutationsite, and a second double-stranded region downstream of said targetmutation site.
 54. A method for detecting mutations in a target DNApolynucleotide, said method comprising: (a) immobilizing a capture probedesigned to hybridize with said target DNA polynucleotide; (b)hybridizing said capture probe to said target DNA polynucleotide,wherein said DNA polynucleotide may contain a mutation relative to anormal or wild type gene; (c) incubating said target polynucleotide withan RNA-polymerase, an initiator, and a terminator; (d) synthesizing anoligonucleotide transcript that is complementary to a target site fromsaid target polynucleotide, wherein said initiator is extended untilsaid terminator is incorporated into said oligonucleotide transcript,thereby synthesizing multiple abortive reiterative oligonucleotidetranscripts; and (e) determining the presence or absence of a mutationby detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts from said target DNA polynucleotide.
 55. Amethod for detecting DNA or RNA in a test sample, said methodcomprising: (a) hybridizing a single stranded target polynucleotide withan abortive promoter cassette comprising a sequence that hybridizes tothe single stranded target polynucleotide, and a region that can bedetected by transcription by a polymerase; (b) incubating said targetpolynucleotide with an RNA-polymerase, an initiator, and a terminator;(c) synthesizing an oligonucleotide transcript that is complementary tothe initiation start site of the APC, wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotides,thereby synthesizing multiple reiterative oligonucleotide transcripts;and (d) detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts.
 56. A method for detecting the presence ofpathogens in a test sample, said method comprising the steps of: (a)hybridizing a single stranded target pathogen polynucleotide in saidtest sample with an abortive promoter cassette comprising a region thatcan be detected by transcription by a polymerase; (b) incubating saidtarget polynucleotide and initiator with an RNA-polymerase, and aterminator; (c) synthesizing an oligonucleotide transcript that iscomplementary to initiation start site of the APC, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple abortive reiterativeoligonucleotide transcripts; and (d) determining the presence of apathogen by detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts synthesized from said test sample.
 57. Themethod of any one of claims 54-56, further comprising detecting orquantifying said reiteratively synthesized oligonucleotide transcript bymodifying a nucleotide in at least one of the members selected from thegroup consisting of said terminator, and said initiator.
 58. The methodof claim 57, wherein said modifying comprises incorporating a labelmoiety.
 59. The method of claim 58, wherein said label moiety comprisesa fluorophore moiety.
 60. The method of claim 59, wherein saidfluorophore moiety comprises a fluorescent energy donor and afluorescent energy acceptor wherein said moiety is detected orquantified by fluorescence resonance energy transfer.
 61. The method ofany one of claims 54-56, wherein said polymerase is selected from thegroup consisting of: a DNA-dependent RNA polymerase, an RNA-dependentRNA polymerase and a modified RNA polymerase, and a primase.
 62. Themethod of claim 61, wherein said polymerase comprises an RNA polymerasederived from one of E. coli, E. coli bacteriophage T7, E. colibacteriophage T3, and S. typhimurium bacteriophage SP6.
 63. The methodof any one of claims 54-56, wherein said abortive oligonucleotides beingsynthesized are one of the lengths selected from the group consistingof: about 2 to about 26 nucleotides, about 26 to about 50 nucleotidesand about 50 nucleotides to about 100 nucleotides.
 64. The method of anyone of claims 54-56, wherein said chain terminator comprises anucleotide analog.
 65. The method of claim 54 or 55, wherein saidinitiator comprises nucleotides selected from the group consisting of:1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250nucleotides, and greater than 250 nucleotides
 66. The method of any oneof claim 56, wherein said single-stranded target polynucleotide is oneof DNA and RNA.
 67. The method of any one of claims 54-56, wherein saidinititator is one of RNA.
 68. The method of claim 56, wherein saidinitiator comprises nucleotides selected from the group consisting of:1-25 nucleotides, 25-50 nucleotides, 50-75 nucleotides, 75-100nucleotides, 100-125 nucleotides, and 125-150 nucleotides, 150-175nucleotides, 175-200 nucleotides, 200-225 nucleotides, and 225-250nucleotides.
 69. The method of claim 55 or claim 56, wherein saidabortive promoter cassette comprises a self-complementaryoligonucleotide that forms a single-stranded bubble in the presence ofan RNA polymerase, wherein a region of said bubble region can bedetected by transcription by said polymerase
 70. The method of any oneof claim 56, wherein said abortive promoter cassette comprises an APClinker which is adapted to hybridize to an end of said target pathogenpolynucleotide.
 71. A method for detecting pathogens in a test sample,said method comprising: (a) immobilizing a capture probe designed tohybridize with a target DNA polynucleotide in said test sample; (b)hybridizing said capture probe with a test sample that potentiallycontains said target DNA polynucleotide; (c) hybridizing a singlestranded target DNA polynucleotide in said test sample with an abortivepromoter cassette comprising a region that hybridizes to the singlestranded target pathogen polynucleotide, and a region that can bedetected by transcription by a polymerase; (d) incubating said targetpolynucleotide with an RNA-polymerase, initiator, and a terminator; (e)synthesizing an oligonucleotide transcript that is complementary to saidinitiation transcription start site of APC, wherein said initiator isextended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple reiterativeoligonucleotide transcripts; and (f) determining the presence or absenceof a pathogen by detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts.
 72. A method for detecting mRNA expressionin a test sample, the method comprising: (a) hybridizing a target mRNAsequence with an abortive promoter cassette comprising a region that canbe detected by transcription by a polymerase; (b) incubating said targetmRNA sequence with an RNA-polymerase, an initiator, and a terminator;(c) synthesizing an oligonucleotide transcript that is complementary totranscription initiation start site, wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotidetranscript, thereby synthesizing multiple reiterative oligonucleotides;and (d) determining the presence or absence of the mRNA by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said test sample.
 73. The method of claim 72, furthercomprising: (a) immobilizing a capture probe, wherein said probehybridizes with a target mRNA sequence; (b) hybridizing said captureprobe with a test sample which potentially contains said target mRNAsequence; and (c) washing a captured target mRNA sequence to removeunhybridized components of said test sample.
 74. The method of claim 72,wherein modifying further comprises incorporating an independentlyselected label moiety into at least one of said initiator, saidterminator, and said nucleotides.
 75. The method of claim 74, whereinsaid label moiety comprises a fluorophore moiety.
 76. The method ofclaim 75, wherein detecting comprises detecting by fluorescenceresonance energy transfer and said fluorophore moiety comprises one of afluorescent energy donor and a fluorescent energy acceptor.
 77. Themethod of claim 72, wherein said polymerase is one of a DNA-dependentRNA polymerase, an RNA-dependent RNA polymerase, an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, and a modified polymerase,and a primase.
 78. The method of claim 72, wherein said polymerasecomprises an RNA polymerase derived from one of E. coli, E. colibacteriophage T7, E. coli bacteriophage T3, and S. typhimuriumbacteriophage SP6.
 79. The method of claim 72, wherein said initiator isone of RNA or DNA.
 80. The method of claim 79, wherein said initiatorcomprises nucleotides selected from the group consisting of: 1-25nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides,101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides,176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, andgreater than 250 nucleotides
 81. The method of claim 72, wherein saidabortive oligonucleotides being synthesized are one of the lengthsselected from the group consisting of: about 2 to about 26 nucleotides,about 26 to about 50 nucleotides and about 50 nucleotides to about 100nucleotides, and greater than 100 nucleotides.
 82. The method of claim72, wherein said abortive promoter cassette comprises aself-complementary oligonucleotide that forms a single-stranded bubbleregion comprising said target site.
 83. The method of claim 72, whereinsaid abortive promoter cassette comprises an APC linker which is adaptedto hybridize to a poly-A tail of said target mRNA sequence.
 84. Themethod of claim 72, wherein said chain terminator comprises one ofnucleotide deprivation and a nucleotide analog.
 85. A method fordetecting an oligonucleotide synthesized from a target DNA sequence, themethod comprising: (a) hybridizing a DNA primer with a single-strandedtarget DNA sequence; (b) extending said DNA primer with a DNA polymeraseand nucleotides, such that said DNA polymerase reiteratively synthesizesa nucleotide sequence; and (c) detecting oligonucleotide comprised ofrepeat sequences synthesized by said DNA polymerase.
 86. The method ofclaim 85, further comprising modifying at least one of said DNA primerand said nucleotides to enable detection of said oligonucleotidecomprised of repeat sequences.
 87. The method of claim 86, whereinmodifying further comprises incorporating an independently selectedlabel moiety into at least one of said DNA primer and said nucleotides.88. The method of claim 87, wherein said label moiety comprises afluorophore moiety.
 89. The method of claim 88, wherein detectingcomprises detecting by fluorescence resonance energy transfer and saidfluorophore moiety comprises one of a fluorescent energy donor and afluorescent energy acceptor.
 90. The method of claim 85, wherein saidDNA polymerase is selected from the group consisting of Escherichia coliDNA polymerase, T7 DNA polymerase, T4 DNA polymerase, Taq thermostableDNA polymerase, terminal transferase, and telomerase.
 91. The method ofclaim 85, wherein said DNA primer comprises from 1 to about 25nucleotides.
 92. The method of claim 85, wherein said oligonucleotiderepeat sequence comprises from about 2 to about 26 nucleotides.
 93. Themethod of claim 85, wherein said detecting comprises hybridizing acomplementary sequence to the synthesized oligonucleotide comprisingrepeat sequences.
 94. The method of claim 93, wherein said complementarysequence is modified to comprise an independently selected label moiety.95. The method of claim 94, wherein said label moiety comprises afluorophore moiety
 96. The method of claim 85, further comprisingimmobilizing said single-stranded target DNA sequence.
 97. The method ofclaim 85, wherein immobilizing comprises hybridizing a capture probe toa portion of said single-stranded target DNA sequence.
 98. The method ofclaim 13, wherein said target site probe and said target DNA sequenceform a bubble complex comprising a first double-stranded region upstreamof said target site, a single-stranded region comprising said targetsite, and a second double-stranded region downstream of said targetsite.
 99. A method of producing a microarray, the method comprising: (a)synthesizing multiple abortive oligonucleotide replicates from a targetDNA sequence by the method of claim 1; and (b) attaching said multipleabortive oligonucleotide replicates to a solid substrate to produce amicroarray of said multiple abortive oligonucleotide replicates. 100.The method of any one of claims 4, 17, 31, 45, 75, 88, or 95 whereinsaid fluorophore moiety is selected from the group consisting of:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)amninonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate; erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1pyrene; butyrate quantum dots; ReactiveRed 4; rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B, sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.
 101. A method fordetecting multiple reiterated oligonucleotides from a target DNA or RNApolynucleotide, said method comprising: (a) incubating a single-strandedtarget polynucleotide in a mixture comprising an initiator, and anRNA-polymerase; (b) synthesizing multiple oligonucleotide transcriptsfrom said target polynucleotide, wherein said initiator is extendeduntil terminated due to nucleotide deprivation, thereby synthesizingmultiple abortive reiterative oligonucleotide transcripts; and (c)detecting or quantifying said reiteratively synthesized oligonucleotides102. A method of detecting multiple reiterated oligonucleotides from atarget DNA or RNA polynucleotide, said method comprising: (a) incubatinga single-stranded target polynucleotide in a mixture comprising aninitiator, an RNA-polymerase, and a target site probe, wherein saidtarget site probe and said target polynucleotide hybridize to form abubble complex comprising a first double-stranded region upstream of atarget site, a single-stranded region comprising said target site, and asecond double-stranded region downstream of said target site; (b)synthesizing multiple oligonucleotide transcripts from said targetpolynucleotide, wherein said initiator is extended until terminated dueto nucleotide deprivation, thereby synthesizing multiple abortivereiterative oligonucleotides; and (c) detecting or quantifying saidreiteratively synthesized oligonucleotide transcripts.
 103. A method fordetecting methylated cytosine residues at a CG site near a target gene,the method comprising: (a) deaminating a single-stranded target DNAsequence under conditions which convert unmethylated cytosine residuesto uracil residues while not converting methylated cytosine residues touracil; (b) incubating a single-stranded target polynucleotide in amixture comprising an initiator, a terminator, an RNA-polymerase, and atarget site probe; (c) synthesizing multiple oligonucleotide transcriptsfrom said target polynucleotide, wherein said initiator is extendeduntil terminated due to nucleotide deprivation, thereby synthesizingmultiple abortive reiterative oligonucleotide transcripts; and (d)detecting or quantifying said reiteratively synthesized oligonucleotides104. The method of claim 26 or 27, further comprising: (a) immobilizingan oligonucleotide capture probe which is specific for a sequence near aCpG island related to a target gene; and (b) hybridizing saidoligonucleotide capture probe with a denatured DNA sample whichpotentially contains said target DNA sequence.
 105. The method of claim27, wherein said target site probe is gene specific cancer specific.106. A method for detecting a target protein in a test sample, themethod comprising: (a) covalently attaching the target protein to anabortive promoter cassette (APC) by a reactive APC linker, wherein saidAPC comprises a region that can be detected by transcription by apolymerase; (b) incubating said target protein with an RNA-polymerase,an initiator, and a terminator; (c) synthesizing an oligonucleotidetranscript that is complementary to transcription initiation start siteof APC, wherein said initiator is extended until said terminator isincorporated into said oligonucleotide transcript, thereby synthesizingmultiple reiterative oligonucleotide transcripts; and (d) determiningthe presence or absence of the target protein by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said test sample.
 107. The method of claim 106 furthercomprising immobilizing target protein by a target specific probe. 108.The method of claim 107, wherein said target specific probe is anantibody.
 109. The method of claim 106, wherein said APC linker will becovalently attached to the target protein by modification ofthiol-reactive or amine-reactive protein crosslinking agents.
 110. Themethod of claim 109 wherein said protein crosslinking agents areselected from the group consisting of: maleamides, iodoacetamides, anddisulfides.
 111. The method of claim 106, wherein said target protein ispurified or in a cell lysate.
 112. A method for detecting cancer,comprising: (a) obtaining a sample from a patient in need of detectionof a cancer; (b) deaminating the DNA under conditions which convertunmethylated cytosine residues to uracil residues while leaving themethylated cytosine residues unaltered; (c) hybridizing an initiator toa target polynucleotide wherein said initiator is a mononucleoside,mononucleotide, binucleotide, oligonucleotide or an analog thereof; (d)incubating said deaminated target polynucleotide and said initiator witha terminator, and an RNA-polymerase, wherein at least one of saidinitiator, terminator is modified to enable detection of the CG sites;(e) synthesizing an oligonucleotide transcript that is complementary tosaid CG sites from said target polynucleotide, wherein said initiator isextended until said terminator is incorporated into said oligonucleotidetranscript thereby synthesizing multiple reiterative oligonucleotidetranscripts; (f) detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts; and comparing the results with thoseobtained similarly from a control sample.
 113. A method for detectingpathogens, said method comprising the steps of: (a) obtaining a samplein need of detection of a pathogen (b) hybridizing a single strandedtarget pathogen polynucleotide in said sample with an abortive promotercassette comprising a nucleotide sequence that hybridizes to singlestranded target pathogen polynucleotide, and a region that can bedetected by transcription by a polymerase; (c) incubating said targetpolynucleotide and initiator with an RNA-polymerase, and a terminator;(d) synthesizing an oligonucleotide transcript that is complementary toinitiation start site of the APC, wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (e) determining the presence of a pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said sample.
 114. The method of claim 113, wherein saidmethod further comprises: (a) immobilizing an oligonucleotide captureprobe which is specific for said target pathogen polynucleotide; and (b)hybridizing said oligonucleotide capture probe with a denatured DNAsample which potentially contains said target pathogen polynucleotide.115. A method for synthesizing multiple reiterated oligonucleotides froma target DNA or RNA polynucleotide, said method comprising: (a)hybridizing an initiator with a single stranded target polynucleotide(b) incubating said target polynucleotide and initiator with anRNA-polymerase, and a terminator; (c) synthesizing multipleoligonucleotides from said target aspolynucleotide, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple reiterativeoligonucleotides.
 116. The method of claim 115, further comprisingsynthesizing oligonucleotides by modifying a nucleotide in at least oneof the members selected from the group consisting of said terminator,and said initiator.
 117. The method of claim 116, wherein said modifyingcomprises incorporating a label moiety.
 118. The method of claim 117,wherein said label moiety comprises a fluorophore moiety.
 119. Themethod of claim 118, wherein said fluorophore moiety comprises afluorescent energy donor and a fluorescent energy acceptor.
 120. Themethod of claim 115, wherein said polymerase is selected from the groupconsisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNApolymerase and a modified RNA-polymerase, and a primase.
 121. The methodof claim 120, wherein said polymerase comprises an RNA polymerasederived from one of E. coli, E. coli bacteriophage T7, E. colibacteriophage T3, and S. typhimurium bacteriophage SP6.
 122. The methodof claim 115, wherein said initiator comprises nucleotides selected fromthe group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225nucleotides, 226-250 nucleotides, and greater than 250 nucleotides 123.The method of claim 115, wherein said abortive oligonucleotides beingsynthesized are one of the lengths selected from the group consistingof: about 2 to about 26 nucleotides, about 26 to about 50 nucleotidesand about 50 nucleotides to about 100 nucleotides.
 124. The method ofclaim 115, wherein said incubating further comprises a target site probespecific for a region on said single-stranded target polynucleotide.125. The method of claim 115, wherein said chain terminator comprises anucleotide analog.
 126. The method of any one of claims 1, 13, 26, 27,41, 54, 55, 56, 71, 72, 101, 102, 103, 106, 112, 113, or 115, whereinsaid incubating further comprises in the presence of ribonucleotides.127. The method of claim 126, wherein said ribonucleotides are modified.128. The method of claim 127, wherein said modifying further comprisesincorporating an independently selected label moiety.
 129. The method ofclaim 128, wherein said label moiety comprises a fluorophore moiety.130. The method of claim 112 or 113, wherein said sample is obtainedfrom the group consisting of: animal, plant or human tissue, blood,saliva, semen, urine, sera, cerebral or spinal fluid, pleural fluid,lymph, sputum, fluid from breast lavage, mucusoal secretions, animalsolids, stool, cultures of microorganisms, liquid and solid food andfeedproducts, waste, cosmetics, air and water.
 131. The method of anyone of claims 55, 56, 71, 72, 106, or 113, wherein said abortivepromoter cassette comprises two partially complementary oligonucleotidesthat form a bubble region.
 132. The method of any one of claims 55, 56,71, 72, 106, or 113, wherein said abortive promoter cassette comprisestwo complementary oligonucleotides that form a bubble region in thepresence of RNA polymerase.
 133. The method of any one of claims 55, 56,71, 72, 106, or 113, wherein said abortive promoter cassette comprisesone contiguous oligonucleotide to which RNA polymerase can bind to forma transcription bubble.
 134. The method of claim 59 wherein saidfluorophore moiety is selected from the group consisting of:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)amninonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate;N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate; erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl lpyrene; butyrate quantum dots; ReactiveRed 4; rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B, sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.
 135. The methodof any one of claims 1, 13, 26, 27, 41, 54-56, 71, 72, 85, 101-103, 106,112, 113, or 115, wherein said initiator is selected from the groupconsisting of: nucleosides, nucleoside analogs, nucleotides, annucleotide analogs.